Inhibitors of C5a for the treatment of viral pneumonia

ABSTRACT

The present invention relates to inhibitors of C5a for use in the treatment of pneumonia, especially viral pneumonia. The invention also relates to the use of inhibitors of C5a in the preparation of a pharmaceutical composition for the treatment of pneumonia, especially viral pneumonia. The inventors further relates to methods for the treatment of pneumonia, especially viral pneumonia, comprising the step of administering a therapeutic amount of an inhibitor of C5a to a subject in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national stage application of InternationalApplication No. PCT/EP2015/055947, filed on Mar. 20, 2015, which claimspriority to European Patent Application 14160947.9, filed on Mar. 20,2014. The disclosures of both European Patent Application 14160947.9 andPCT/EP2015/055947 are hereby incorporated herein by reference in theirentireties.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readable aminoacid/nucleotide sequence listing submitted concurrently herewith andidentified as follows: One 8,749 byte ASCII (Text) file named“70854-258331_SL.txt” created on Sep. 14, 2016.

The present invention relates to inhibitors of C5a for use in thetreatment of pneumonia, especially viral pneumonia. The invention alsorelates to the use of inhibitors of C5a in the preparation of apharmaceutical composition for the treatment of pneumonia, especiallyviral pneumonia. The inventors further relates to methods for thetreatment of pneumonia, especially viral pneumonia, comprising the stepof administering a therapeutic amount of an inhibitor of C5a to asubject in need thereof.

BACKGROUND OF THE INVENTION

C5a

C5a is cleaved from C5 upon complement activation. Among the complementactivation products, C5a is one of the most potent inflammatorypeptides, with a broad spectrum of functions (Guo R F, and Ward P A.2005. Annu. Rev. Immunol. 23:821-852). C5a is a glycoprotein present inthe blood of healthy humans with a molecular weight of 11.2 kDa. Thepolypeptide portion of C5a contains 74 amino acids, accounting for amolecular weight of 8.2 kDa while the carbohydrate portion accounts forapproximately 3 kDa. C5a exerts its effects through the high-affinityC5a receptors (C5aR and C5L2) (Ward P A. 2009. J. Mol. Med.87(4):375-378). C5aR belongs to the rhodopsin-type family ofG-protein-coupled receptors with seven transmembrane segments; C5L2 issimilar but is not G-protein-coupled. It is currently believed that C5aexerts its biological functions primarily through C5a-C5aR interaction,as few biological responses have been found for C5a-C5L2 interaction.C5aR is widely expressed on myeloid cells including neutrophils,eosinophils, basophils, and monocytes, and non-myeloid cells in manyorgans, especially in the lung and liver, indicative of the importanceof C5a/C5aR signaling. C5a has a variety of biological functions (Guoand Ward, 2005, supra). C5a is a strong chemoattractant for neutrophilsand also has chemotactic activity for monocytes and macrophages. C5acauses an oxidative burst (O₂ consumption) in neutrophils and enhancesphagocytosis and release of granular enzymes. C5a has also been found tobe a vasodilator. C5a has been shown to be involved in modulation ofcytokine expression from various cell types, to enhance expression ofadhesion molecules on neutrophils. It is found that C5a becomes highlydetrimental when it is overly produced in the disease settings, as it isa strong inducer and enhancer for inflammatory responses functioning inthe up-stream of the inflammatory reaction chain. High doses of C5a canlead to nonspecific chemotactic “desensitization” for neutrophils,thereby causing broad dysfunction (Huber-Lang M et al. 2001. J. Immunol.166(2):1193-1199).

C5a has been reported to exert numerous pro-inflammatory responses. Forexample, C5a stimulates the synthesis and release from human leukocytesof pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-8, andmacrophage migration inhibitory factor (MIF) (Hopken U et al. 1996. EurJ Immunol 26(5):1103-1109; Riedemann N C et al. 2004. J Immunol173(2):1355-1359; Strieter R M et al. 1992. Am J Pathol 141(2):397-407).C5a produces a strong synergistic effect with LPS in production ofTNF-α, macrophage inflammatory protein (MIP)-2, cytokine-inducedneutrophil chemoattractant (CINC)-1, and IL-1β in alveolar epithelialcells (Riedemann N C et al. 2002. J. Immunol. 168(4):1919-1925;Rittirsch D et al. 2008. Nat Rev Immunol 8(10):776-787).

Blockade of C5a has also been proven to be protective in experimentalmodels of sepsis and in many other models of inflammation such asischemia/reperfusion injury, renal disease, graft rejection, malaria,rheumatoid arthritis, infectious bowel disease, inflammatory lungdisease, lupus-like auto-immune diseases, neurodegenerative disease,etc. in various species as partially reviewed under Klos A. et al (KlosA. et al. 2009. Mol Immunol 46(14):2753-2766) and Allegretti M. et al(Allegretti M et al. 2005. Curr Med Chem 12(2):217-236). Moreover, ithas been recently discovered that blockade of C5a has shown a strongtherapeutic benefit in a tumor model in mice (Markiewski M M et al.2008. Nat Immunol 9(11): 1225-1235).

Avian Influenza

A novel avian influenza H7N9 virus emerged in China in February 2013 anda total of 139 patients with 45 fatal cases were confirmed till November2013 (WHO. Human infection with avian influenza A(H7N9) virus—update.http://www.who.int/csr/don/2013_11_06/en/index.html (accessed on Nov.16, 2013)). Most severe cases infected with H7N9 viral infection hadmanifestation of viral pneumonia with acute lung injury (ALI) and thenprogressed to severe respiratory failure and acute respiratory distresssyndrome (ARDS) which was similar to the pathogenesis in patientsinfected with HPAI (highly pathogenic avian influenza) H5N1 virus orsevere acute respiratory syndrome (SARS) virus (Beigel J H et al. 2005.N Engl J Med 353:1374-1385; Ip W K, et al. 2005. J Infect Dis191:1697-1704). To date, no therapeutic strategies have been found toeffectively treat these diseases. Accumulating studies suggested thatthe complement activation occurred in severe patients infected withinfluenza virus and was closely associated with the levels ofproinflammatory mediators and lung injury. It has been reported thatpatients with severe pdmH1NI (pandemic influenza H1N1) virus infectionhad strong systemic complement activation with increased production ofproinflammatory mediators (Berdal J E et al. 2011. J. Infect.63(4):308-16; Ohta R et al. 2011. Microbiol. Immunol. 55(3):191-8). Inaddition, our previous studies have showed that the complementactivation products in lung tissue sections and plasma samples werelargely increased in the mouse model of H5N1 infection, and that thepathogenesis of ALI could be attributable, at least in part, to thecomplement activation and associated activation products such as C3a andC5a (Sun, S. et al. 2013. Am J Respir Cell Mol Biol 49: 221-230).

Complement system is a central part of the immune system in hostdefenses against pathogen invasion and in clearance of potentiallydamaging cell debris. However, excessive complement activation could bedetrimental, since it may contribute to uncontrolled inflammatoryresponses and lead to tissue damages (Daniel Ricklin & John D Lambris.2013 J Immunol 190(8):3831-8). Complement has become an interesting andpromising target for treatment of various clinical diseases such asischemia/reperfusion (I/R) injury, transplantation and autoimmunedisorders (Lu F. et al. 2013. Cardiovasc. Pathol. 22:75-80; Tillou, X.et al. 2010. Kidney Int. 78:152-159; Manderson A P, et al. 2004. AnnuRev Immunol 22:431-456. Since the role of complement activation in theoutcome of pathogen-induced diseases could be more complex due to thediversity of pathogen biological features including propagation andpathogenicity as well as a potential “dual role” of complementactivation in the pathogen-driven immune responses, it is important toconsider preservation of pathogen clearance function while inhibitinginflammation and tissue injury for the development of complementinhibitors for the treatment of pathogen-associated inflammatorydisorders.

Complement activation product C5a exerts a predominant proinflammatoryactivity and mediates strong proinflammatory and modulatory signals inmany disease models (Klos A. et al. 2009, supra). To date, manytherapeutic compounds targeting C5a or C5aR such as C5a inhibitor C5aIP,C5aR antagonist PMX53 and CCX168 had been tested in the preclinicalmodels with promising therapeutic benefits in transplantation, sepsis,arthritis, renal vasculitis and cancer (Woodruff, T. M. et al. 2011.Mol. Immunol. 48:1631-1642; Okada, N. et al. 2012. Clin. Exp. Pharmacol.2:114; Tokodai, K. et al. 2010. Transplantation 90:1358-1365; Köhl, J.2006. Curr. Opin. Mol. Ther. 8: 529-538). It was also demonstrated thatantibody blockade of C5a or C5a receptor abrogated the excessive immuneresponses in the mouse model of Plasmodium berghei ANKA (PbA) infection(Patel, S. N. et al. 2008. J. Exp. Med. 205:1133-1143). Similarly, ourprevious study employing a mouse model of HPAI H5N1 viral pneumoniarevealed that anti-C5a treatment significantly attenuated lung injuryand improved the survival rate (Sun, S. et al. 2013, supra). Sincemembrane attack complex (MAC) plays an essential role in the innate hostdefenses again invading pathogens, it appears to be advantageous toapply C5a blockade strategy inhibiting the inflammatory responsesderived from pathogen infection while leaving the arm of MAC formationintact.

Technical Problems Underlying the Present Invention

One of the problems underlying the invention was the provision oftherapeutic approaches for the treatment of viral pneumonia, inparticular for the treatment of viral pneumonia caused by the novelavian influenza H7N9 virus.

So far it has not been studied whether an anti-C5a treatment would beeffective in the treatment of viral pneumonia caused by the novel avianinfluenza virus H7N9. Previous studies are focused on other avianinfluenza viruses (H5N1; cf. Sun, S. et al. 2013, supra) and onlyemployed a mouse model to study viral pneumonia caused by the avianinfluenza virus. Positive results from a mouse model might not always betransferable to an actual treatment of human patients.

The inventors of the instant application have applied IFX-1, a highlypotent neutralizing mAb against human C5a, which is currently in theclinical development, in a monkey model of H7N9 virus infection toexplore the therapeutic potential of complement inhibition in thetreatment of H7N9 virus-induced severe pneumonia. To our knowledge, thisis the first time that an anti-C5a treatment of viral pneumonia has beenstudied in a monkey model.

The data in the experimental section shown below demonstrate thatexcessive complement activation occurs in the H7N9 infection and it isattributable to the development of ALI (acute lung injury) and systemicinflammation.

The present inventors have found that anti-C5a treatment in H7N9infected monkeys substantially attenuated ALI and led to stronglyreduced lung histopathologic injury scores as well as decreased lunginfiltration of macrophages and neutrophils when compared to untreatedinfected African green monkeys. In addition, the intensity of theinfectious SIRS (systemic inflammatory response syndrome) caused by H7N9was markedly reduced by IFX-1 treatment, as evidenced by a significantreduction in body temperature increases and in plasma levels ofinflammatory mediators. The virus titers in the infected lungs of AGMswere unexpectedly diminished by IFX-1 treatment. This is verysurprising, since there is no known mechanism which would suggest aninvolvement of C5a in virus replication.

The results suggest that complement inhibition is a highly promisingstrategy for an adjunctive treatment of severe viral pneumonia. Thetherapeutic effects associated with the administration of a C5ainhibitor are so preeminent that even a monotherapy for viral pneumoniabased on a C5a inhibitor appears to be feasible.

The above overview does not necessarily describe all advantagesassociated with and problems solved by the present invention.

SUMMARY OF THE INVENTION

In a first aspect the present invention relates to an inhibitor of C5afor use in the reduction of viral load and/or reduction of acute lunginjury (ALI) in a subject suffering from viral pneumonia.

In a second aspect the present invention relates to an inhibitor of C5afor use in the treatment of pneumonia (preferably viral pneumonia) in asubject, wherein the inhibitor is for use as a monotherapy.

In a third aspect the present invention relates to an inhibitor of C5afor use in the treatment of viral pneumonia in a subject, wherein theviral pneumonia in the subject is caused by an H7N9 virus.

In a fourth aspect the present invention relates to an inhibitor of C5afor use in the treatment of pneumonia (preferably viral pneumonia) in asubject, wherein the subject is a primate, preferably an ape, morepreferably a human.

This summary of the invention does not necessarily describe all featuresof the invention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Before the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodology, protocols and reagents described herein as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, Leuenberger, H. G. W, Nagel, B. and Kölbl, H. eds.(1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

Several documents (for example: patents, patent applications, scientificpublications, manufacturer's specifications, instructions, GenBankAccession Number sequence submissions etc.) are cited throughout thetext of this specification. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention. Some of the documents cited herein arecharacterized as being “incorporated by reference”. In the event of aconflict between the definitions or teachings of such incorporatedreferences and definitions or teachings recited in the presentspecification, the text of the present specification takes precedence.

Sequences: All sequences referred to herein are disclosed in theattached sequence listing that, with its whole content and disclosure,is a part of this specification.

As used herein, “human C5a” refers to the following 74 amino acidpeptide:

(SEQ ID NO: 1) TLQKKIEEIA AKYKHSVVKK CCYDGACVNN DETCEQRAARISLGPRCIKA FTECCVVASQ LRANISHKDM QLGR.As used herein, the term “human C5a” refers to glycosylated forms and todeglycosylated forms of this 74 amino acid peptide. The terms “humanC5a” and “hC5a” are used interchangeably herein.

The term “inhibitor of C5a”, as used herein, refers to a compound thatinhibits a biological activity of C5a. The term “inhibitor of C5a”particularly refers to a compound that interferes with the binding ofC5a to the C5a receptors, C5aR and C5L2; especially to a compound thatinterferes with the binding of C5a to C5aR. Accordingly, the term“inhibitor of C5a” encompasses compounds that specifically bind to C5aand inhibit binding of C5a to C5aR as well as compounds thatspecifically bind to C5aR and inhibit binding of C5a to C5aR. Exemplaryinhibitors of C5a include the C5a inhibitory peptide (C5aIP), theselective C5a receptor antagonists PMX53 and CCX168, and the anti-C5aantibodies disclosed in WO 2011/063980 A1 (also published as US2012/0231008 A1). The term “inhibitor of C5a” and “C5a inhibitor” areused interchangeably herein.

The term “binding moiety”, as used herein, refers to any molecule orpart of a molecule that can specifically bind to a target molecule ortarget epitope. Preferred binding moieties in the context of the presentapplication are (a) antibodies or antigen-binding fragments thereof; (b)oligonucleotides; (c) antibody-like proteins; or (d) peptidomimetics.Exemplary “binding moieties” that are especially well-suited forpracticing the present invention are capable of binding to aconformational epitope of human C5a which is formed by the two aminoacid sequences NDETCEQRA (SEQ ID NO: 2) and SHKDMQL (SEQ ID NO: 3).Further exemplary “binding moieties” that are especially well-suited forpracticing the present invention are capable of binding to aconformational epitope of human C5a which is formed by the two aminoacid sequences DETCEQR (SEQ ID NO: 4) and HKDMQ (SEQ ID NO: 5).

As used herein, a first compound (e.g. an antibody) is considered to“bind” to a second compound (e.g. an antigen, such as a target protein),if it has a dissociation constant K_(d) to said second compound of 1 mMor less, preferably 100 μM or less, preferably 50 μM or less, preferably30 μM or less, preferably 20 μM or less, preferably 10 μM or less,preferably 5 μM or less, more preferably 1 μM or less, more preferably900 nM or less, more preferably 800 nM or less, more preferably 700 nMor less, more preferably 600 nM or less, more preferably 500 nM or less,more preferably 400 nM or less, more preferably 300 nM or less, morepreferably 200 nM or less, even more preferably 100 nM or less, evenmore preferably 90 nM or less, even more preferably 80 nM or less, evenmore preferably 70 nM or less, even more preferably 60 nM or less, evenmore preferably 50 nM or less, even more preferably 40 nM or less, evenmore preferably 30 nM or less, even more preferably 20 nM or less, andeven more preferably 10 nM or less.

The term “binding” according to the invention preferably relates to aspecific binding. “Specific binding” means that a binding moiety (e.g.an antibody) binds stronger to a target such as an epitope for which itis specific compared to the binding to another target. A binding moietybinds stronger to a first target compared to a second target if it bindsto the first target with a dissociation constant (K_(d)) which is lowerthan the dissociation constant for the second target. Preferably thedissociation constant (K_(d)) for the target to which the binding moietybinds specifically is more than 10-fold, preferably more than 20-fold,more preferably more than 50-fold, even more preferably more than100-fold, 200-fold, 500-fold or 1000-fold lower than the dissociationconstant (K_(d)) for the target to which the binding moiety does notbind specifically.

As used herein, the term “K_(d)” (usually measured in “mol/L”, sometimesabbreviated as “M”) is intended to refer to the dissociation equilibriumconstant of the particular interaction between a binding moiety (e.g. anantibody or fragment thereof) and a target molecule (e.g. an antigen orepitope thereof). In the context of the present application, the “K_(d)”value is determined by surface plasmon resonance spectroscopy (Biacore™)at room temperature (25° C.).

An “epitope”, also known as antigenic determinant, is the part of amacromolecule that is recognized by the immune system, specifically byantibodies, B cells, or T cells. As used herein, an “epitope” is thepart of a macromolecule capable of binding to a binding moiety (e.g. anantibody or antigen-binding fragment thereof) as described herein. Inthis context, the term “binding” preferably relates to a specificbinding. Epitopes usually consist of chemically active surface groupingsof molecules such as amino acids or sugar side chains and usually havespecific three-dimensional structural characteristics, as well asspecific charge characteristics. Conformational and non-conformationalepitopes can be distinguished in that the binding to the former but notthe latter is lost in the presence of denaturing solvents.

As used herein, a “conformational epitope” refers to an epitope of alinear macromolecule (e.g. a polypeptide) that is formed by thethree-dimensional structure of said macromolecule. In the context of thepresent application, a “conformational epitope” is a “discontinuousepitope”, i.e. the conformational epitope on the macromolecule (e.g. apolypeptide) which is formed from at least two separate regions in theprimary sequence of the macromolecule (e.g. the amino acid sequence of apolypeptide). In other words, an epitope is considered to be a“conformational epitope” in the context of the present invention, if theepitope consists of at least two separate regions in the primarysequence to which a binding moiety of the invention (e.g. an antibody oran antigen-binding fragment thereof) binds simultaneously, wherein theseat least two separate regions are interrupted by one or more regions inthe primary sequence to which a binding moiety of the invention does notbind. Preferably, such a “conformational epitope” is present on apolypeptide, and the two separate regions in the primary sequence aretwo separate amino acid sequences to which a binding moiety of theinvention (e.g. an antibody or an antigen-binding fragment thereof)binds, wherein these at least two separate amino acid sequences areinterrupted by one or more amino acid sequences in the primary sequenceto which a binding moiety of the invention does not bind. Preferably,the interrupting amino acid sequence is a contiguous amino acid sequencecomprising two or more amino acids to which the binding moiety does notbind. The at least two separate amino acid sequences to which a bindingmoiety of the invention binds are not particularly limited with regardto their length. Such a separate amino acid sequence may consists ofonly one amino acid as long as the total number of amino acids withinsaid at least two separate amino acid sequences is sufficiently large toeffect specific binding between the binding moiety and theconformational epitope.

A “paratope” is the part of an antibody that binds to the epitope. Inthe context of the present invention, a “paratope” is the part of abinding moiety (e.g. an antibody or antigen-binding fragment thereof) asdescribed herein that binds to the epitope.

The term “antibody” typically refers to a glycoprotein comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen-binding portion thereof. The term“antibody” also includes all recombinant forms of antibodies, inparticular of the antibodies described herein, e.g. antibodies expressedin prokaryotes, unglycosylated antibodies, antibodies expressed ineukaryotes (e.g. CHO cells), glycosylated antibodies, and anyantigen-binding antibody fragments and derivatives as described below.Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as VH or V_(H)) and a heavy chain constant region(abbreviated herein as CH or C_(H)). The heavy chain constant region canbe further subdivided into three parts, referred to as CH1, CH2, and CH3(or C_(H)1, C_(H)2, and C_(H)3). Each light chain is comprised of alight chain variable region (abbreviated herein as VL or V_(L)) and alight chain constant region (abbreviated herein as CL or C_(L)). The VHand VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavyand light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (C1q) of the classical complement system.

The term “antigen-binding fragment” of an antibody (or simply “bindingportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen. Ithas been shown that the antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) Fab fragments, monovalent fragments consisting ofthe VL, VH, CL and CH domains; (ii) F(ab′)₂ fragments, bivalentfragments comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) Fd fragments consisting of the VH and CHdomains; (iv) Fv fragments consisting of the VL and VH domains of asingle arm of an antibody, (v) dAb fragments (Ward et al., (1989) Nature341: 544-546), which consist of a VH domain; (vi) isolatedcomplementarity determining regions (CDR), and (vii) combinations of twoor more isolated CDRs which may optionally be joined by a syntheticlinker. Furthermore, although the two domains of the Fv fragment, VL andVH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding fragment” ofan antibody. A further example is a binding-domain immunoglobulin fusionprotein comprising (i) a binding domain polypeptide that is fused to animmunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavychain CH2 constant region fused to the hinge region, and (iii) animmunoglobulin heavy chain CH3 constant region fused to the CH2 constantregion. The binding domain polypeptide can be a heavy chain variableregion or a light chain variable region. The binding-domainimmunoglobulin fusion proteins are further disclosed in US 2003/0118592and US 2003/0133939. These antibody fragments are obtained usingconventional techniques known to those skilled in the art, and thefragments are screened for utility in the same manner as are intactantibodies. Further examples of “antigen-binding fragments” areso-called microantibodies, which are derived from single CDRs. Forexample, Heap et al., 2005, describe a 17 amino acid residuemicroantibody derived from the heavy chain CDR3 of an antibody directedagainst the gp120 envelope glycoprotein of HIV-1 (Heap C. J. et al.(2005) Analysis of a 17-amino acid residue, virus-neutralizingmicroantibody. J. Gen. Virol. 86:1791-1800). Other examples includesmall antibody mimetics comprising two or more CDR regions that arefused to each other, preferably by cognate framework regions. Such asmall antibody mimetic comprising V_(H) CDR1 and V_(L) CDR3 linked bythe cognate V_(H) FR2 has been described by Qiu et al., 2007 (Qiu X.-Q.et al. (2007) Small antibody mimetics comprising twocomplementary-determining regions and a framework region for tumortargeting. Nature biotechnology 25(8):921-929).

Thus, the term “antibody or antigen-binding fragment thereof”, as usedherein, refers to immunoglobulin molecules and immunologically activeportions of immunoglobulin molecules, i.e. molecules that contain anantigen-binding site that immunospecifically binds an antigen. Alsocomprised are immunoglobulin-like proteins that are selected throughtechniques including, for example, phage display to specifically bind toa target molecule or target epitope, e.g. to the conformational epitopeof human C5a formed by the amino acid sequences according to SEQ ID NO:2 and SEQ ID NO: 3; or the conformational epitope of human C5a formed bythe amino acid sequences DETCEQR (SEQ ID NO: 4) and HKDMQ (SEQ ID NO:5). The immunoglobulin molecules of the invention can be of any type(e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2,preferably IgG2a and IgG2b, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule.

Antibodies and antigen-binding fragments thereof usable in the inventionmay be from any animal origin including birds and mammals. Preferably,the antibodies or fragments are from human, chimpanzee, rodent (e.g.mouse, rat, guinea pig, or rabbit), chicken, turkey, pig, sheep, goat,camel, cow, horse, donkey, cat, or dog origin. It is particularlypreferred that the antibodies are of human or murine origin. Antibodiesof the invention also include chimeric molecules in which an antibodyconstant region derived from one species, preferably human, is combinedwith the antigen binding site derived from another species, e.g. mouse.Moreover, antibodies of the invention include humanized molecules inwhich the antigen binding sites of an antibody derived from a non-humanspecies (e.g. from mouse) are combined with constant and frameworkregions of human origin.

As exemplified herein, antibodies of the invention can be obtaineddirectly from hybridomas which express the antibody, or can be clonedand recombinantly expressed in a host cell (e.g., a CHO cell, or alymphocytic cell). Further examples of host cells are microorganisms,such as E. coli, and fungi, such as yeast. Alternatively, they can beproduced recombinantly in a transgenic non-human animal or plant.

The term “chimeric antibody” refers to those antibodies wherein oneportion of each of the amino acid sequences of heavy and light chains ishomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular class, while theremaining segment of the chain is homologous to corresponding sequencesin another species or class. Typically the variable region of both lightand heavy chains mimics the variable regions of antibodies derived fromone species of mammals, while the constant portions are homologous tosequences of antibodies derived from another. One clear advantage tosuch chimeric forms is that the variable region can conveniently bederived from presently known sources using readily available B-cells orhybridomas from non-human host organisms in combination with constantregions derived from, for example, human cell preparations. While thevariable region has the advantage of ease of preparation and thespecificity is not affected by the source, the constant region beinghuman is less likely to elicit an immune response in a human subjectwhen the antibodies are injected than would the constant region from anon-human source. However, the definition is not limited to thisparticular example.

The term “humanized antibody” refers to a molecule having anantigen-binding site that is substantially derived from animmunoglobulin from a non-human species, wherein the remainingimmunoglobulin structure of the molecule is based upon the structureand/or sequence of a human immunoglobulin. The antigen-binding site mayeither comprise complete variable domains fused onto constant domains oronly the complementarity determining regions (CDR) grafted ontoappropriate framework regions in the variable domains. Antigen-bindingsites may be wild-type or modified by one or more amino acidsubstitutions, e.g. modified to resemble human immunoglobulins moreclosely. Some forms of humanized antibodies preserve all CDR sequences(for example a humanized mouse antibody which contains all six CDRs fromthe mouse antibody). Other forms have one or more CDRs which are alteredwith respect to the original antibody.

Different methods for humanizing antibodies are known to the skilledperson, as reviewed by Almagro & Fransson, 2008, Frontiers inBioscience, 13:1619-1633, the content of which is herein incorporated byreference in its entirety. The review article by Almagro & Fransson isbriefly summarized in US 2012/0231008 A1 which is the national stageentry of international patent application WO 2011/063980 A1. Thecontents of US 2012/0231008 A1 and WO 2011/063980 A1 are hereinincorporated by reference in their entirety.

As used herein, “human antibodies” include antibodies having variableand constant regions derived from human germline immunoglobulinsequences. The human antibodies of the invention may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo). Human antibodies of the invention includeantibodies isolated from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and that do not expressendogenous immunoglobulins, as described for example in U.S. Pat. No.5,939,598 by Kucherlapati & Jakobovits.

The term “monoclonal antibody” as used herein refers to a preparation ofantibody molecules of single molecular composition. A monoclonalantibody displays a single binding specificity and affinity for aparticular epitope. In one embodiment, the monoclonal antibodies areproduced by a hybridoma which includes a B cell obtained from anon-human animal, e.g. mouse, fused to an immortalized cell.

The term “recombinant antibody”, as used herein, includes all antibodiesthat are prepared, expressed, created or isolated by recombinant means,such as (a) antibodies isolated from an animal (e.g., a mouse) that istransgenic or transchromosomal with respect to the immunoglobulin genesor a hybridoma prepared therefrom, (b) antibodies isolated from a hostcell transformed to express the antibody, e.g. from a transfectoma, (c)antibodies isolated from a recombinant, combinatorial antibody library,and (d) antibodies prepared, expressed, created or isolated by any othermeans that involve splicing of immunoglobulin gene sequences to otherDNA sequences.

The term “transfectoma”, as used herein, includes recombinant eukaryotichost cells expressing an antibody, such as CHO cells, NS/0 cells, HEK293cells, HEK293T cells, plant cells, or fungi, including yeast cells.

As used herein, a “heterologous antibody” is defined in relation to atransgenic organism producing such an antibody. This term refers to anantibody having an amino acid sequence or an encoding nucleic acidsequence corresponding to that found in an organism not consisting ofthe transgenic organism, and being generally derived from a speciesother than the transgenic organism.

As used herein, a “heterohybrid antibody” refers to an antibody havinglight and heavy chains of different organismal origins. For example, anantibody having a human heavy chain associated with a murine light chainis a heterohybrid antibody.

Thus, “antibodies and antigen-binding fragments thereof” suitable foruse in the present invention include, but are not limited to,polyclonal, monoclonal, monovalent, bispecific, heteroconjugate,multispecific, recombinant, heterologous, heterohybrid, chimeric,humanized (in particular CDR-grafted), deimmunized, or human antibodies,Fab fragments, Fab′ fragments, F(ab′)₂ fragments, fragments produced bya Fab expression library, Fd, Fv, disulfide-linked Fvs (dsFv), singlechain antibodies (e.g. scFv), diabodies or tetrabodies (Holliger P. etal. (1993) Proc. Natl. Acad. Sci. U.S.A. 90(14), 6444-6448), nanobodies(also known as single domain antibodies), anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above.

The antibodies described herein are preferably isolated. An “isolatedantibody” as used herein, is intended to refer to an antibody which issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds to C5ais substantially free of antibodies that specifically bind antigensother than C5a). An isolated antibody that specifically binds to anepitope, isoform or variant of human C5a may, however, havecross-reactivity to other related antigens, e.g. from other species(e.g. C5a species homologs, such as rat C5a). Moreover, an isolatedantibody may be substantially free of other cellular material and/orchemicals. In one embodiment of the invention, a combination of“isolated” monoclonal antibodies relates to antibodies having differentspecificities and being combined in a well-defined composition.

The term “naturally occurring”, as used herein, as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally occurring.

As used herein, the term “nucleic acid aptamer” refers to a nucleic acidmolecule that has been engineered through repeated rounds of in vitroselection or SELEX (systematic evolution of ligands by exponentialenrichment) to bind to a target molecule (for a review see: Brody E. N.and Gold L. (2000), Aptamers as therapeutic and diagnostic agents. J.Biotechnol. 74(1):5-13). The nucleic acid aptamer may be a DNA or RNAmolecule. The aptamers may contain modifications, e.g. modifiednucleotides such as 2′-fluorine-substituted pyrimidines.

As used herein, the term “antibody-like protein” refers to a proteinthat has been engineered (e.g. by mutagenesis of loops) to specificallybind to a target molecule. Typically, such an antibody-like proteincomprises at least one variable peptide loop attached at both ends to aprotein scaffold. This double structural constraint greatly increasesthe binding affinity of the antibody-like protein to levels comparableto that of an antibody. The length of the variable peptide loop istypically between 10 and 20 amino acids. The scaffold protein may be anyprotein having good solubility properties. Preferably, the scaffoldprotein is a small globular protein. Antibody-like proteins includewithout limitation affibodies, anticalins, and designed ankyrin repeatproteins (for review see: Binz H. K. et al. (2005) Engineering novelbinding proteins from nonimmunoglobulin domains. Nat. Biotechnol.23(10):1257-1268). Antibody-like proteins can be derived from largelibraries of mutants, e.g. be panned from large phage display librariesand can be isolated in analogy to regular antibodies. Also,antibody-like binding proteins can be obtained by combinatorialmutagenesis of surface-exposed residues in globular proteins.Antibody-like proteins are sometimes referred to as “peptide aptamers”.

As used herein, a “peptidomimetic” is a small protein-like chaindesigned to mimic a peptide. Peptidomimetics typically arise frommodification of an existing peptide in order to alter the molecule'sproperties. For example, they may arise from modifications to change themolecule's stability or biological activity. This can have a role in thedevelopment of drug-like compounds from existing peptides. Thesemodifications involve changes to the peptide that will not occurnaturally (such as altered backbones and the incorporation of nonnaturalamino acids).

“Conservative substitutions” may be made, for instance, on the basis ofsimilarity in polarity, charge, size, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the amino acid residuesinvolved. Amino acids can be grouped into the following six standardamino acid groups:

(1) hydrophobic: Met, Ala, Val, Leu, Be;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro; and

(6) aromatic: Trp, Tyr, Phe.

As used herein, “conservative substitutions” are defined as exchanges ofan amino acid by another amino acid listed within the same group of thesix standard amino acid groups shown above. For example, the exchange ofAsp by Glu retains one negative charge in the so modified polypeptide.In addition, glycine and proline may be substituted for one anotherbased on their ability to disrupt α-helices. Some preferred conservativesubstitutions within the above six groups are exchanges within thefollowing sub-groups: (i) Ala, Val, Leu and Ile; (ii) Ser and Thr; (iii)Asn and Gln; (iv) Lys and Arg; and (v) Tyr and Phe. Given the knowngenetic code, and recombinant and synthetic DNA techniques, the skilledscientist readily can construct DNAs encoding the conservative aminoacid variants.

As used herein, “non-conservative substitutions” or “non-conservativeamino acid exchanges” are defined as exchanges of an amino acid byanother amino acid listed in a different group of the six standard aminoacid groups (1) to (6) shown above.

As used herein, the expression “comprises 1, 2, or 3 amino acidexchanges, preferably conservative amino acid exchanges, 1, 2, or 3amino acid deletions, and/or 1, 2, or 3 amino acid additions” has to beunderstood in that the so modified amino acid sequence contains no morethan 3 amino acid exchanges (preferably conservative amino acidexchanges), no more than 3 amino acid deletions, and no more than 3amino acid additions. Consequently, the thus characterized amino acidsequence has a maximum of 9 amino acid modifications (3 exchanges+3deletions+3 additions). Accordingly, the afore-mentioned expression is aclosed expression, even though the term “comprises” is understood as anopen expression in other contexts.

A “biological activity” as used herein, refers to any activity apolypeptide may exhibit, including without limitation: enzymaticactivity; binding activity to another compound (e.g. binding to anotherpolypeptide, in particular binding to a receptor, or binding to anucleic acid); inhibitory activity (e.g. enzyme inhibitory activity);activating activity (e.g. enzyme-activating activity); or toxic effects.Regarding variants and derivatives of a polypeptide, it is not requiredthat the variant or derivative exhibits such an activity to the sameextent as the parent polypeptide. A variant is regarded as a variantwithin the context of the present application, if it exhibits therelevant activity to a degree of at least 10% (e.g. at least 20%, atleast 30%, at least 40%, or at least 50%) of the activity of the parentpolypeptide. Likewise, a derivative is regarded as a derivative withinthe context of the present application, if it exhibits the relevantbiological activity to a degree of at least 10% of the activity of theparent polypeptide. A particularly relevant “biological activity” in thecontext of the present invention is a binding activity to theconformational epitope of human C5a formed by the amino acid sequencesaccording to SEQ ID NO: 2 and SEQ ID NO: 3. Preferably, the relevant“biological activity” in the context of the present invention is abinding activity to the conformational epitope of human C5a formed bythe amino acid sequences DETCEQR (SEQ ID NO: 4) and KDM. Assays fordetermining binding activity are known to a person of ordinary skill inthe art and include ELISA and surface plasmon resonance assays.

As used herein, a “patient” means any mammal or bird who may benefitfrom a treatment with an inhibitor of C5a described herein. Preferably,a “patient” is selected from the group consisting of laboratory animals(e.g. mouse or rat), domestic animals (including e.g. guinea pig,rabbit, chicken, turkey, pig, sheep, goat, camel, cow, horse, donkey,cat, or dog), or primates including monkeys (e.g. African green monkeys,chimpanzees, bonobos, gorillas) and human beings. It is particularlypreferred that the “patient” is a human being. The terms “patient” and“subject to be treated” (or just: “subject”) are used interchangeablyherein.

As used herein, the term “monkey” refers to any non-human primatemammal, if the context does not say otherwise. For example, in thesection “Examples” below, the term “monkey” is typically used as anabbreviation for “African green monkey”.

As used herein, the term “ape” refers to Old World anthropoid mammalsbelonging to the biological superfamily Hominoidea and, accordingly,includes gibbons (family Hilobatidae), orang-utans (genus Pongo),gorillas (genus Gorilla), chimpanzees (genus Pan), and humans (genusHomo).

As used herein, “treat”, “treating” or “treatment” of a disease ordisorder means accomplishing one or more of the following: (a) reducingthe severity and/or duration of the disorder; (b) limiting or preventingdevelopment of symptoms characteristic of the disorder(s) being treated;(c) inhibiting worsening of symptoms characteristic of the disorder(s)being treated; (d) limiting or preventing recurrence of the disorder(s)in patients that have previously had the disorder(s); and (e) limitingor preventing recurrence of symptoms in patients that were previouslysymptomatic for the disorder(s).

As used herein, “prevent”, “preventing”, “prevention”, or “prophylaxis”of a disease or disorder means preventing that a disorder occurs in asubject for a certain amount of time. For example, if an inhibitor ofC5a described herein (e.g. an anti-C5a antibody or an antigen-bindingfragment thereof) is administered to a subject with the aim ofpreventing a disease or disorder, said disease or disorder is preventedfrom occurring at least on the day of administration and preferably alsoon one or more days (e.g. on 1 to 30 days; or on 2 to 28 days; or on 3to 21 days; or on 4 to 14 days; or on 5 to 10 days) following the day ofadministration.

As used herein, “administering” includes in vivo administration, as wellas administration directly to tissue ex vivo, such as vein grafts.

A “pharmaceutical composition” according to the invention may be presentin the form of a composition, wherein the different active ingredientsand diluents and/or carriers are admixed with each other, or may takethe form of a combined preparation, where the active ingredients arepresent in partially or totally distinct form. An example for such acombination or combined preparation is a kit-of-parts.

An “effective amount” is an amount of a therapeutic agent sufficient toachieve the intended purpose. The effective amount of a giventherapeutic agent will vary with factors such as the nature of theagent, the route of administration, the size and species of the subjectto receive the therapeutic agent, and the purpose of the administration.The effective amount in each individual case may be determinedempirically by a skilled artisan according to established methods in theart.

As used herein, the term “adjunctive therapy” refers to a combinationtherapy, in which at least two different drugs are administered to thepatient. These at least two different drugs can be formulated into onesingle pharmaceutical composition containing both drugs. Alternatively,each drug can be formulated into a separate pharmaceutical compositionand the pharmaceutical compositions are separately administered (e.g. atdifferent time-points and/or by different routes of administration) tothe patient. In this latter alternative, the (at least) two differentdrugs can be present in a kit-of-parts. The present disclosureparticularly features a therapy with a C5a inhibitor as an adjunctivetherapy to antiviral treatment with an antiviral agent.

As used herein, the term “antiviral agent” includes without limitation:neuraminidase inhibitors (e.g. orally inhaled zanamivir or oraloseltamivir) and virus-specific antibodies.

“Pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

The term “carrier”, as used herein, refers to a diluent, adjuvant,excipient, or vehicle with which the therapeutic agent is administered.Such pharmaceutical carriers can be sterile liquids, such as salinesolutions in water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. A saline solution is a preferred carrierwhen the pharmaceutical composition is administered intravenously.Saline solutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice flour, chalk, silica gel, sodium stearate,glycerol mono stearate, talc, sodium chloride, dried skim milk,glycerol, propylene glycol, water, ethanol and the like. Thecomposition, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents. These compositions can takethe form of solutions, suspensions, emulsions, tablets, pills, capsules,powders, sustained-release formulations and the like. The compositioncan be formulated as a suppository, with traditional binders andcarriers such as triglycerides. The compounds of the invention can beformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with free amino groups such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with free carboxyl groups such as those derived fromsodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine,triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. Examplesof suitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

Generally known and practiced methods in the fields of molecularbiology, cell biology, protein chemistry and antibody techniques arefully described in the continuously updated publications “MolecularCloning: A Laboratory Manual”, (Sambrook et al., Cold Spring Harbor);Current Protocols in Molecular Biology (F. M. Ausubel et al. Eds., Wiley& Sons); Current Protocols in Protein Science (J. E. Colligan et al.Eds., Wiley & Sons); Current Protocols in Cell Biology (J. S. Bonifacinoet al., Wiley & Sons) and Current Protocols in Immunology (J. E.Colligan et al., Eds., Wiley & Sons). Known techniques relating to cellculture and media are described in “Large Scale Mammalian Cell Culture(D. Hu et al., Curr. Opin. Biotechnol. 8:148-153, 1997); “Serum freeMedia” (K. Kitano, Biotechnol. 17:73-106, 1991); and “Suspension Cultureof Mammalian Cells” (J. R. Birch et al. Bioprocess Technol. 10:251-270,1990).

Embodiments of the Invention

The present invention will now be further described. In the followingpassages different aspects of the invention are defined in more detail.Each aspect defined below may be combined with any other aspect oraspects unless clearly indicated to the contrary. In particular, anyfeature indicated as being preferred or advantageous may be combinedwith any other feature or features indicated as being preferred oradvantageous.

In a first aspect the present invention is directed to an inhibitor ofC5a for use in the reduction of viral load and/or reduction of acutelung injury (ALI) in a subject suffering from viral pneumonia,especially HxNx-mediated viral pneumonia.

In an alternative wording, the first aspect of the present invention isdirected to the use of an inhibitor of C5a in the preparation of apharmaceutical composition for the reduction of viral load and/orreduction of acute lung injury (ALI) in a subject suffering from viralpneumonia, especially HxNx-mediated viral pneumonia.

In another alternative wording, the first aspect of the presentinvention is directed to a method for the reduction of viral load and/orreduction of acute lung injury (ALI) in a subject suffering from viralpneumonia, especially HxNx-mediated viral pneumonia, said methodcomprising the step of administering a therapeutic amount of aninhibitor of C5a to said subject.

In a second aspect the present invention is directed to an inhibitor ofC5a for use in the treatment of pneumonia (preferably viral pneumonia,especially HxNx-mediated viral pneumonia) in a subject, wherein theinhibitor is for use as a monotherapy.

In an alternative wording, the second aspect of the present invention isdirected to the use of an inhibitor of C5a in the preparation of apharmaceutical composition for the treatment of pneumonia (preferablyviral pneumonia, especially HxNx-mediated viral pneumonia) in a subject,wherein the pharmaceutical composition is to be used as a monotherapy.

In another alternative wording, the second aspect of the presentinvention is directed to a method for the treatment of pneumonia(preferably viral pneumonia, especially HxNx-mediated viral pneumonia),said method comprising the step of administering a therapeutic amount ofan inhibitor of C5a to a subject in need thereof, wherein the inhibitoris administered as a monotherapy.

In a third aspect the present invention is directed to an inhibitor ofC5a for use in the treatment of viral pneumonia in a subject, whereinthe viral pneumonia in the subject is caused by an H7N9 virus.

In an alternative wording, the third aspect of the present invention isdirected to the use of an inhibitor of C5a in the preparation of apharmaceutical composition for the treatment of viral pneumonia in asubject, wherein the viral pneumonia in the subject is caused by an H7N9virus.

In another alternative wording, the third aspect of the presentinvention is directed to a method for the treatment of viral pneumonia,said method comprising the step of administering a therapeutic amount ofan inhibitor of C5a to a subject in need thereof, wherein the viralpneumonia in said subject is caused by an H7N9 virus.

In a fourth aspect the present invention is directed to an inhibitor ofC5a for use in the treatment of pneumonia (preferably viral pneumonia,especially HxNx-mediated viral pneumonia) in a subject, wherein thesubject is a primate, preferably an ape, more preferably a human.

In an alternative wording, the fourth aspect of the present invention isdirected to the use of an inhibitor of C5a in the preparation of apharmaceutical composition for the treatment of pneumonia (preferablyviral pneumonia, especially HxNx-mediated viral pneumonia) in a subject,wherein the subject is a primate, preferably an ape, more preferably ahuman.

In another alternative wording, the fourth aspect of the presentinvention is directed to a method for the treatment of pneumonia(preferably viral pneumonia, especially HxNx-mediated viral pneumonia),said method comprising the step of administering a therapeutic amount ofan inhibitor of C5a to a subject in need thereof, wherein said subjectis a primate, preferably an ape, more preferably a human.

In one embodiment of the second, third, or fourth aspect of theinvention, the inhibitor of C5a is for use in the reduction of viralload and/or acute lung injury (ALI) in a subject suffering from viralpneumonia or—in an alternative wording—the pharmaceutical composition isfor the reduction of viral load and/or reduction of acute lung injury(ALI) in a subject suffering from viral pneumonia or—in anotheralternative wording—the method is for reduction of viral load and/oracute lung injury (ALI) in a subject suffering from viral pneumonia.

In one embodiment of the first, third, or fourth aspect of theinvention, the inhibitor of C5a is for use as a monotherapy or—in analternative wording—the pharmaceutical composition is to be used as amonotherapy or—in another alternative wording—the inhibitor of C5a isadministered as a monotherapy.

In another embodiment of the first, third or fourth aspect of theinvention, the inhibitor of C5a is for use in an adjunctive therapy withan antiviral agent or—in an alternative wording—the pharmaceuticalcomposition is to be used in an adjunctive therapy with an antiviralagent or—in another alternative wording—the method additionallycomprises the step of administering a therapeutic amount of an antiviralagent to said subject. Antiviral agents suitable for use in such anadjunctive therapy include without limitation: neuraminidase inhibitors(e.g. orally inhaled zanamivir or oral oseltamivir) and virus-specificantibodies.

In one embodiment of the first, second, or fourth aspect of theinvention, the pneumonia in the subject is caused by an HxNx virus. Insome embodiments, the HxNx virus is selected from the group consistingof H1N1, H1N3, H2N2, H3N2, H5N1, H7N2, H7N3, H7N7, H7N9, H9N2, H10N7,and H10N8. In particular embodiments of the first, second, or fourthaspect of the invention, the HxNx virus is H7N9.

In one embodiment of the first, second, or third aspect of theinvention, the subject is a primate, preferably an ape, more preferablya human.

The present invention further provides combinations of the four aspectsdefined above. For example, in one embodiment, the features of thefirst, second, and third aspect of the invention can be combined. Inanother embodiment, the features of the first, second, and fourth aspectof the invention can be combined. In another embodiment, the features ofthe first, third, and fourth aspect of the invention can be combined. Inanother embodiment, the features of the second, third, and fourth aspectof the invention can be combined. In yet another embodiment, thefeatures of the first, second, third, and fourth aspect of the inventioncan be combined.

In one embodiment of the first, second, third, or fourth aspect of theinvention, the inhibitor of C5a is selected from the group consisting of(i) compounds (binding moieties) that specifically bind to C5a andinhibit binding of C5a to C5aR; and (ii) compounds (binding moieties)that specifically bind to C5aR and inhibit binding of C5a to C5aR.Exemplary compounds that specifically bind to C5a include the C5ainhibitory peptide (C5aIP) and anti-C5a antibodies, such as the anti-C5aantibodies disclosed in WO 2011/063980 A1 (also published as US2012/0231008 A1). Exemplary compounds that specifically bind to C5aRinclude the selective C5a receptor antagonists PMX53 and CCX168.

In one embodiment of the first, second, third, or fourth aspect of theinvention, the inhibitor of C5a is a binding moiety specifically bindingto human C5a. In a further embodiment, said binding moiety is selectedfrom the group consisting of

-   (a) antibodies or antigen-binding fragments thereof;-   (b) oligonucleotides;-   (c) antibody-like proteins; and-   (d) peptidomimetics.

In one embodiment of the first, second, third, or fourth aspect of theinvention, the binding moiety specifically binds to a conformationalepitope formed by amino acid sequences NDETCEQRA (SEQ ID NO: 2) andSHKDMQL (SEQ ID NO: 3) of human C5a. Binding to the conformationalformed by the amino acid sequences according to SEQ ID NOs: 2 and 3means that the binding moiety binds to at least one amino acid withinthe amino acid sequence according to SEQ ID NO: 2 and to at least oneamino acid within the amino acid sequence according to SEQ ID NO: 3. SEQID NO: 2 corresponds to amino acids 30-38 of human C5a. SEQ ID NO: 3corresponds to amino acids 66-72 of human C5a.

In preferred embodiments of the first, second, third, or fourth aspectof the invention, the binding moiety binds to at least one amino acidwithin the amino acid sequence according to DETCEQR (SEQ ID NO: 4). SEQID NO: 4 corresponds to amino acids 31-37 of human C5a.

In further preferred embodiments of the first, second, third, or fourthaspect of the invention, the binding moiety binds to at least one aminoacid within the amino acid sequence according to HKDMQ (SEQ ID NO: 5),more preferably to at least one amino acid within the amino acidsequence KDM. SEQ ID NO: 5 corresponds to amino acids 67-71 of humanC5a; the sequence KDM corresponds to amino acids 68-70 of human C5a.

In preferred embodiments of the first, second, third, or fourth aspectof the invention, the binding moiety binds to at least one amino acidwithin the amino acid sequence DETCEQR (SEQ ID NO: 4) and to at leastone amino acid within the amino acid sequence HKDMQ (SEQ ID NO: 5).

In particularly preferred embodiments of the first, second, third, orfourth aspect of the invention, the binding moiety binds to at least oneamino acid within the amino acid sequence DETCEQR (SEQ ID NO: 4) and toat least one amino acid within the amino acid sequence KDM.

In preferred embodiments of the first, second, third, or fourth aspectof the invention, the two sequences forming the conformational epitope(e.g. sequence pairs according to SEQ ID NO: 2 and 3; SEQ ID NO: 4 and5; or SEQ ID NO: 4 and sequence KDM) are separated by 1-50 contiguousamino acids that do not participate in binding to the binding moiety ofthe invention. In the following, such amino acids that do notparticipate in binding to the binding moiety of the invention will bereferred to as “non-binding amino acids”. The two sequences forming theconformational epitope are preferably separated by 6-45 contiguousnon-binding amino acids, more preferably by 12-40 contiguous non-bindingamino acids, more preferably by 18-35 contiguous non-binding aminoacids, more preferably by 24-30 contiguous non-binding amino acids, morepreferably by 25-29 contiguous non-binding amino acids, even morepreferably by 26-28 contiguous non-binding amino acids, and mostpreferably by 27 contiguous non-binding amino acids.

In preferred embodiments of the first, second, third, or fourth aspectof the invention, the binding moiety has a binding constant to human C5awith a K_(d) value of 10 nM or less, preferably 9 nM or less, morepreferably 8 nM or less, more preferably 7 nM or less, more preferably 6nM or less, more preferably 5 nM or less, more preferably 4 nM or less,more preferably 3 nM or less, more preferably 2 nM or less, and evenmore preferably 1 nM or less.

In preferred embodiments of the first, second, third, or fourth aspectof the invention, the dissociation constant K_(d) between the bindingmoiety and human C5a is between 1 pM (picomolar) and 5 nM (nanomolar),more preferably between 2 pM and 4 nM, more preferably between 5 pM and3 nM, more preferably between 10 pM and 2 nM, more preferably between 50pM and 1 nM, more preferably between 100 pM and 900 pM, more preferablybetween 200 pM and 800 pM, more preferably between 300 pM and 700 pM,and even more preferably between 400 pM and 600 pM.

In preferred embodiments of the first, second, third, or fourth aspectof the invention, one binding moiety exhibits at least 75% blockingactivity, preferably at least 80% blocking activity, more preferably atleast 85% blocking activity, more preferably at least 90% blockingactivity, more preferably at least 95% blocking activity for biologicaleffects induced by one molecule C5a, particularly human C5a. Thesepreferred blocking activities refer to those embodiments, wherein thebinding moiety comprises a single paratope binding to C5a, preferablyhuman C5a. In embodiments, wherein the binding moiety comprises two ormore C5a-specific paratopes, said blocking activities of at least 75%,preferably at least 80%, more preferably at least 85%, etc. are achievedwhen one binding-moiety molecule is contacted with a number of C5amolecules equal to the number of C5a-specific paratopes present in thebinding moiety. In other words, when the paratopes of a binding moietydescribed herein and C5a are present in equimolar concentrations, thebinding moiety exhibits at least 75% blocking activity, preferably atleast 80% blocking activity, more preferably at least 85% blockingactivity, more preferably at least 90% blocking activity, and morepreferably at least 95% blocking activity for biological effects inducedby C5a. A preferred biological effect to be blocked is C5a-inducedlysozyme release from human whole blood cells. Assays for determiningthis C5a-induced lysozyme release and its blocking are described, forexample, in WO 2011/063980 A1.

In preferred embodiments of the first, second, third, or fourth aspectof the invention, the binding moiety does not inhibit CH50 activity inhuman plasma. Assays for determining CH50 activity are known to theskilled person and are described, for example, in WO 2011/063980 A1.

In preferred embodiments of the first, second, third, or fourth aspectof the invention, the binding moiety does not exhibit a blockingactivity on at least one C5b-induced biological effect; preferably thebinding moiety does not exhibit a blocking activity on any C5b-inducedbiological effect.

In preferred embodiments of the first, second, third, or fourth aspectof the invention, the binding moiety is capable of reducing E. coliinduced IL-8 production in human whole blood. Assays for measuring IL-8production in whole blood are known to the skilled person and aredescribed, for example, in WO 2011/063980 A1.

In preferred embodiments of the first, second, third, or fourth aspectof the invention, the binding moiety is an antibody, said antibody beingselected from the group consisting of polyclonal antibodies, monoclonalantibodies, monovalent antibodies, bispecific antibodies,heteroconjugate antibodies, multispecific antibodies, deimmunizedantibodies, chimeric antibodies, humanized (in particular CDR-grafted)antibodies, and human antibodies.

In preferred embodiments of the first, second, third, or fourth aspectof the invention, the binding moiety is an antigen-binding fragment ofan antibody, said fragment being selected from the group consisting ofFab fragments, Fab′ fragments, F(ab′)₂ fragments, Fd fragments, Fvfragments, disulfide-linked Fvs (dsFv), single domain antibodies (alsoknown as nanobodies), and single chain Fv (scFv) antibodies.

In one embodiment of the first, second, third or fourth aspect of theinvention, the binding moiety is an antibody or an antigen-bindingfragment thereof, wherein said antibody or antigen-binding fragmentthereof comprises

-   (i) a heavy chain CDR3 sequence as set forth in SEQ ID NO: 6; or-   (ii) a heavy chain CDR3 sequence as set forth in SEQ ID NO: 7;    wherein the heavy chain CDR3 sequence optionally comprises 1, 2, or    3 amino acid exchanges, preferably conservative amino acid    exchanges, 1, 2, or 3 amino acid deletions, and/or 1, 2, or 3 amino    acid additions.

In one embodiment of the first, second, third or fourth aspect of theinvention, the binding moiety is an antibody or an antigen-bindingfragment thereof, wherein said antibody or antigen-binding fragmentthereof comprises

-   (iii) a light chain CDR3 sequence as set forth in SEQ ID NO: 8; or-   (iv) a light chain CDR3 sequence as set forth in SEQ ID NO: 9;    wherein the light chain CDR3 sequence optionally comprises 1, 2, or    3 amino acid exchanges, preferably conservative amino acid    exchanges, 1, 2, or 3 amino acid deletions, and/or 1, 2, or 3 amino    acid additions.

In some embodiments of the first, second, third or fourth aspect of theinvention, the binding moiety is an antibody or an antigen-bindingfragment thereof, wherein said antibody or antigen-binding fragmentthereof comprises

-   (i) a heavy chain CDR3 sequence as set forth in SEQ ID NO: 6 and a    light chain CDR3 sequence as set forth in SEQ ID NO: 8; or-   (ii) a heavy chain CDR3 sequence as set forth in SEQ ID NO: 7 and a    light chain CDR3 sequence as set forth in SEQ ID NO: 9;    wherein the heavy chain CDR3 sequence optionally comprises 1, 2, or    3 amino acid exchanges, preferably conservative amino acid    exchanges, 1, 2, or 3 amino acid deletions, and/or 1, 2, or 3 amino    acid additions; and    wherein the light chain CDR3 sequence optionally comprises 1, 2, or    3 amino acid exchanges, preferably conservative amino acid    exchanges, 1, 2, or 3 amino acid deletions, and/or 1, 2, or 3 amino    acid additions.

In one embodiment of the first, second, third or fourth aspect of theinvention, the binding moiety is an antibody or an antigen-bindingfragment thereof, wherein said antibody or antigen-binding fragmentthereof comprises at least one of the following sequences:

-   (v) a heavy chain CDR2 sequence according to SEQ ID NO: 10;-   (vi) a heavy chain CDR2 sequence according to SEQ ID NO: 11;-   (vii) a light chain CDR2 sequence according to SEQ ID NO: 12;-   (viii) a light chain CDR2 sequence according to SEQ ID NO: 13;-   (ix) a heavy chain CDR1 sequence according to SEQ ID NO: 14;-   (x) a heavy chain CDR1 sequence according to SEQ ID NO: 15;-   (xi) a light chain CDR1 sequence according to SEQ ID NO: 16; or-   (xii) a light chain CDR1 sequence according to SEQ ID NO: 17;    wherein the heavy chain CDR2 sequence optionally comprises 1, 2, or    3 amino acid exchanges, preferably conservative amino acid    exchanges, 1, 2, or 3 amino acid deletions, and/or 1, 2, or 3 amino    acid additions;    wherein the light chain CDR2 sequence optionally comprises 1, 2, or    3 amino acid exchanges, preferably conservative amino acid    exchanges, 1, 2, or 3 amino acid deletions, and/or 1, 2, or 3 amino    acid additions;    wherein the heavy chain CDR1 sequence optionally comprises 1, 2 or 3    amino acid exchanges, preferably conservative amino acid exchanges,    1, 2, or 3 amino acid deletions, and/or 1, 2, or 3 amino acid    additions; and    wherein the light chain CDR1 sequence optionally comprises 1, 2, or    3 amino acid exchanges, preferably conservative amino acid    exchanges, 1, 2, or 3 amino acid deletions, and/or 1, 2, or 3 amino    acid additions.

Preferably, the total number of these optional changes recited above ineach one of the amino acid sequences according to SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17,i.e. the total number of exchanges, deletions and additions in eachsequence, is 1 or 2.

Preferably the total number of exchanges, deletions, and additions addedup for all CDRs present in an antibody or antigen-binding fragmentthereof is between 1 and 5 (e.g. 1, 2, 3, 4, or 5).

In preferred embodiments of the first, second, third or fourth aspect ofthe invention, the antibody or antigen-binding fragment thereofcomprises one of the sets of heavy chain CDR3, heavy chain CDR2, andheavy chain CDR1 sequences as listed below in Table 1,

wherein each heavy chain CDR3 sequence optionally comprises 1, 2, or 3amino acid exchanges, preferably conservative amino acid exchanges, 1,2, or 3 amino acid deletions, and/or 1, 2, or 3 amino acid additions;

wherein each heavy chain CDR2 sequence optionally comprises 1, 2, or 3amino acid exchanges, preferably conservative amino acid exchanges, 1,2, or 3 amino acid deletions, and/or 1, 2, or 3 amino acid additions;and

wherein each heavy chain CDR1 sequence optionally comprises 1, 2, or 3amino acid exchanges, preferably conservative amino acid exchanges, 1,2, or 3 amino acid deletions, and/or 1, 2, or 3 amino acid additions:

TABLE 1 Sets of heavy chain CDR sequences suitable for use in theantibodies or fragments thereof of the present invention Symbol of heavyCDR3 chain set sequence CDR2 sequence CDR1 sequence A SEQ ID NO: 6 SEQID NO: 10 SEQ ID NO: 14 B SEQ ID NO: 6 SEQ ID NO: 10 SEQ ID NO: 15 C SEQID NO: 6 SEQ ID NO: 11 SEQ ID NO: 14 D SEQ ID NO: 6 SEQ ID NO: 11 SEQ IDNO: 15 E SEQ ID NO: 7 SEQ ID NO: 10 SEQ ID NO: 14 F SEQ ID NO: 7 SEQ IDNO: 10 SEQ ID NO: 15 G SEQ ID NO: 7 SEQ ID NO: 11 SEQ ID NO: 14 H SEQ IDNO: 7 SEQ ID NO: 11 SEQ ID NO: 15

In preferred embodiments of the first, second, third or fourth aspect ofthe invention, the antibody or antigen-binding fragment thereofcomprises one of the following sets of light chain CDR3, light chainCDR2, and light chain CDR1 sequences as listed in Table 2,

wherein each light chain CDR3 sequence optionally comprises 1, 2, or 3amino acid exchanges, preferably conservative amino acid exchanges, 1,2, or 3 amino acid deletions, and/or 1, 2, or 3 amino acid additions;

wherein each light chain CDR2 sequence optionally comprises 1, 2, or 3amino acid exchanges, preferably conservative amino acid exchanges, 1,2, or 3 amino acid deletions, and/or 1, 2, or 3 amino acid additions;and

wherein each light chain CDR1 sequence optionally comprises 1, 2, or 3amino acid exchanges, preferably conservative amino acid exchanges, 1,2, or 3 amino acid deletions, and/or 1, 2, or 3 amino acid additions:

TABLE 2 Sets of light chain CDR sequences suitable for use in theantibodies or fragments thereof of the present invention Since the CDR2light chain sequence of antibody IFX-1 (SEQ ID NO: 12) is identical tothe CDR2 light chain sequence of antibody INab708 (SEQ ID NO: 13), setsincluding SEQ ID NO: 13 would be redundant to sets including SEQ ID NO:12. Therefore, the table only list four sets of light chain CDRsequences. Number of light CDR3 chain set sequence CDR2 sequence CDR1sequence I SEQ ID NO: 8 SEQ ID NO: 12 SEQ ID NO: 16 II SEQ ID NO: 8 SEQID NO: 12 SEQ ID NO: 17 III SEQ ID NO: 9 SEQ ID NO: 12 SEQ ID NO: 16 IVSEQ ID NO: 9 SEQ ID NO: 12 SEQ ID NO: 17In preferred embodiments of the first, second, third or fourth aspect ofthe invention, the antibody or antigen-binding fragment thereofcomprises one of the heavy CDR sets A-H listed above in Table 1 and oneof the light chain CDR sets I-IV listed above in Table 2, i.e. one ofthe following combinations of sets: A-I, A-II, A-III, A-IV, B-I, B-II,B-III, B-IV, C-I, C-II, C-III, C-IV, D-I, D-II, D-III, D-IV, E-I, E-II,E-III, E-IV, F-I, F-II, F-III, F-IV, G-I, G-II, G-III, G-IV, H-I, H-II,H-III, or H-IV (wherein the combinations A-I and H-IV are especiallypreferred),wherein each heavy chain CDR3 sequence optionally comprises 1, 2, or 3amino acid exchanges, preferably conservative amino acid exchanges, 1,2, or 3 amino acid deletions, and/or 1, 2, or 3 amino acid additions;wherein each heavy chain CDR2 sequence optionally comprises 1, 2, or 3amino acid exchanges, preferably conservative amino acid exchanges, 1,2, or 3 amino acid deletions, and/or 1, 2, or 3 amino acid additions;wherein each heavy chain CDR1 sequence optionally comprises 1, 2, or 3amino acid exchanges, preferably conservative amino acid exchanges, 1,2, or 3 amino acid deletions and/or 1, 2, or 3 amino acid additions;wherein each light chain CDR3 sequence optionally comprises 1, 2, or 3amino acid exchanges, preferably conservative amino acid exchanges, 1,2, or 3 amino acid deletions, and/or 1, 2, or 3 amino acid additions;wherein each light chain CDR2 sequence optionally comprises 1, 2, or 3amino acid exchanges, preferably conservative amino acid exchanges, 1,2, or 3 amino acid deletions, and/or 1, 2, or 3 amino acid additions;andwherein each light chain CDR1 sequence optionally comprises 1, 2, or 3amino acid exchanges, preferably conservative amino acid exchanges, 1,2, or 3 amino acid deletions and/or 1, 2, or 3 amino acid additions.

In preferred embodiments of the first, second, third or fourth aspect ofthe invention, the antibody or antigen-binding fragment thereofcomprises a VH domain that comprises, essentially consists of orconsists of (i) the VH domain of IFX-1 or (ii) the VH domain of INab708.

The FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 sequences defining the VHdomains of IFX-1 and INab708 are shown below in Table 3.

In preferred embodiments of the first, second, third or fourth aspect ofthe invention, the antibody or antigen-binding fragment thereofcomprises a VL domain that comprises, essentially consists of orconsists of (i) the VL domain of IFX-1 or (ii) the VL domain of INab708.

The FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 sequences defining the VLdomains of IFX-1 and INab708 are shown below in Table 3.

TABLE 3 CDR and FR sequences of antibodies IFX-1 and  INab708 (Chothia classification mode) IFX-1: INab708: Heavy Chain:Heavy Chain: FR1:  FR1:  QVQLQQSGPQLVRPGTSVKIS VQLLESGAELMKPGASVKIS (=SEQ ID NO: 18) (SEQ ID NO: 26) CDR1: CKASGYSFTTFWMD CDR1: CKATGNTFSGYWIE(= SEQ ID NO: 14) (= SEQ ID NO: 15) FR2: WVKQRPGQGLEWIGRFR2: WVKQRPGHGLEWIGE (SEQ ID NO: 19) (SEQ ID NO: 27) CDR2: IDPSDSESRLDQCDR2: ILPGSGSTNYNE (= SEQ ID NO: 10) (= SEQ ID NO: 11) FR3: FR3:RFKDRATLTVDKSSSTVYMQLSSP KFKGKATLTADTSSNTAYMQLSSL TSEDSAVYY TSEDSAVYY(SEQ ID NO: 20) (SEQ ID NO: 28) CDR3: CARGNDGYYGFAYCDR3: CTRRGLYDGSSYFAY (= SEQ ID NO: 6) (= SEQ ID NO: 7) FR4: WGQGTLVTVSSFR4: WGQGTLVTVSA (SEQ ID NO: 21) (SEQ ID NO: 29) Light chain:Light Chain: FR1:  FR1:  DIVLTQSPASLAVSLGQRATIS DIVLTQSPASLAVSLGQRATIS(SEQ ID NO: 22) (SEQ ID NO: 30) CDR1: CKASQSVDYDGDSYMKCDR1: CKASQSVDYDGDSYMN (= SEQ ID NO: 16) (= SEQ ID NO: 17)FR2: WYQQKPGQPPKLL FR2: WYQQKPGQPPKLL (SEQ ID NO: 23) (SEQ ID NO: 31)CDR2: IYAASNL CDR2: IYAASNL (= SEQ ID NO: 12) (= SEQ ID NO: 13) FR3:FR3: QSGIPARFSGSGSGTDFTLNIHPV GSGIPARFSGSGSGTDFTLNIHPV EEEDAATYYEEEVAATYY (SEQ ID NO: 24) (SEQ ID NO: 32) CDR3: CQQSNEDPYTCDR3: CQQNNEDPLT (= SEQ ID NO: 8) (= SEQ ID NO: 9) FR4: FGGGTKLEIKFR4: FGAGTLLELK (SEQ ID NO: 25) (SEQ ID NO: 33)

As will be further explained below in the section “Examples”, IFX-1 is achimeric human/mouse monoclonal IgG4 antibody developed by InflaRx GmbH,Germany. IFX-1 is derived from mouse monoclonal antibody INab308described in WO 2011/063980 A1. IFX-1 has the same heavy chain variableregion and the same light chain variable region as INab308. INab708 is amouse monoclonal antibody targeting essentially the same conformationalepitope as INab308 and IFX-1 and is also described in WO 2011/063980 A1.

In further preferred embodiments of the first, second, third or fourthaspect of the invention, the antibody or antigen-binding fragmentthereof comprises a VH domain and a VL domain, wherein

-   -   (i) said VH domain comprises, essentially consists of or        consists of the VH domain of IFX-1 and said VL domain comprises,        essentially consists of or consists of the VL domain of IFX-1;        or    -   (ii) said VH domain comprises, essentially consists of or        consists of the VH domain of INab708 and said VL domain        comprises, essentially consists of or consists of the VL domain        of INab708.

In preferred embodiments of the first, second, third or fourth aspect ofthe invention, the antibody or antigen-binding fragment thereofcomprising one or more CDRs, a set of CDRs or a combination of sets ofCDRs as described herein comprises said CDRs in a human antibodyframework.

Reference herein to an antibody comprising with respect to the heavychain thereof a particular chain, or a particular region or sequencepreferably relates to the situation wherein all heavy chains of saidantibody comprise said particular chain, region or sequence. Thisapplies correspondingly to the light chain of an antibody.

In some embodiments of the first, second, third or fourth aspect of theinvention, the binding moiety is an oligonucleotide. In theseembodiments, it is further preferred that the oligonucleotide is anucleic acid aptamer, such as a DNA aptamer or RNA aptamer or a mixedaptamer comprising DNA and RNA nucleotides. In some embodiments, one ormore nucleotides may be replaced by modified nucleotides such as2′-fluorine-substituted pyrimidines. Nucleic acid aptamers may also beconjugated with fluorescent reporter molecules, affinity tags and/ormacromolecules. For example, conjugating the aptamer topolyethylenglycol (PEG) or to a comparable macromolecule will increasethe biological half-life of the aptamer.

In some embodiments of the first, second, third or fourth aspect of theinvention, the binding moiety is an antibody-like protein, e.g. anantibody-like protein as exemplified above in the “Definitions” section.

In some embodiments of the first, second, third or fourth aspect of theinvention, the binding moiety is a peptidomimetic. Peptidomimeticssuitable for practicing the present invention are preferably based onantibody-like proteins as described above.

The teaching given herein with respect to specific nucleic acid andamino acid sequences, e.g. those shown in the sequence listing, is to beconstrued so as to also relate to modifications of said specificsequences resulting in sequences which are functionally equivalent tosaid specific sequences, e.g. amino acid sequences exhibiting propertiesidentical or similar to those of the specific amino acid sequences andnucleic acid sequences encoding amino acid sequences exhibitingproperties identical or similar to those of the amino acid sequencesencoded by the specific nucleic acid sequences. One important propertyis to retain binding of an antibody to its target or to sustain effectorfunctions of an antibody. Preferably, a sequence modified with respectto a specific sequence, when it replaces the specific sequence in anantibody retains binding of said antibody to C5a, in particular to theconformational epitope of C5a identified herein, and preferably retainsfunctions of said antibody as described herein, e.g. blockingC5a-induced lysozyme release from human whole blood cells and/orreducing E. coli induced IL-8 production in human whole blood.

It will be appreciated by those skilled in the art that in particularthe sequences of the CDR hypervariable and variable regions can bemodified without losing the ability to bind C5a. For example, CDRregions will be either identical or highly homologous to the regionsspecified herein. By “highly homologous” it is contemplated that from 1to 5, preferably from 1 to 4, such as 1 to 3 or 1 or 2 exchanges, inparticular conservative exchanges, deletions, and/or additions may bemade in the CDRs. In addition, the hypervariable and variable regionsmay be modified so that they show substantial homology with the regionsspecifically disclosed herein.

Furthermore, it may be desired according to the present invention tomodify the amino acid sequences described herein, in particular those ofhuman heavy chain constant regions to adapt the sequence to a desiredallotype, e.g. an allotype found in the Caucasian population or in theChinese population.

The present invention further comprises antibodies in which alterationshave been made in the Fc region in order to change the functional orpharmacokinetic properties of the antibodies. Such alterations mayresult in a decrease or increase of C1q binding and CDC or of FcγRbinding and ADCC (antibody-dependent cellular cytotoxicity).Substitutions can, for example, be made in one or more of the amino acidresidues of the heavy chain constant region, thereby causing analteration in an effector function while retaining the ability to bindto the antigen as compared with the modified antibody, cf. U.S. Pat. No.5,624,821 and U.S. Pat. No. 5,648,260.

The in vivo half-life of antibodies can be improved by modifying thesalvage receptor epitope of the Ig constant domain or an Ig-likeconstant domain such that the molecule does not comprise an intact CH2domain or an intact Ig Fc region, cf. U.S. Pat. No. 6,121,022 and U.S.Pat. No. 6,194,551. The in vivo half-life can furthermore be increasedby making mutations in the Fc region, e.g., by substituting threoninefor leucine at position 252, by substituting threonine for serine atposition 254, or by substituting threonine for phenylalanine at position256, cf. U.S. Pat. No. 6,277,375.

Furthermore, the glycosylation pattern of antibodies can be modified inorder to change the effector function of the antibodies. For example,the antibodies can be expressed in a transfectoma which does not add thefucose unit normally attached to Asn at position 297 of the Fc region inorder to enhance the affinity of the Fc region for Fc-Receptors which,in turn, will result in an increased ADCC (antibody-dependent cellularcytotoxicity) of the antibodies in the presence of NK cells, cf. Shieldet al. (2002) J. Biol. Chem., 277:26733-40. Furthermore, modification ofgalactosylation can be made in order to modify CDC (complement-dependentcytotoxicity).

Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of an anti-C5a antibody coding sequence, suchas by saturation mutagenesis, and the resulting modified anti-C5aantibodies can be screened for binding activity.

In the practice of any aspect of the present invention, a pharmaceuticalcomposition as described herein or an inhibitor of C5a (e.g. a bindingmoiety specifically binding to C5a, especially hC5a, as describedherein) may be administered to a patient by any route established in theart which provides a sufficient level of the inhibitor of C5a in thepatient. It can be administered systemically or locally. Suchadministration may be parenterally, transmucosally, e.g., orally,nasally, rectally, intravaginally, sublingually, submucosally,transdermally, or by inhalation. Preferably, administration isparenteral, e.g., via intravenous or intraperitoneal injection, and alsoincluding, but is not limited to, intra-arterial, intramuscular,intradermal and subcutaneous administration. If the pharmaceuticalcomposition of the present invention is administered locally it can beinjected directly into the organ or tissue to be treated.

Pharmaceutical compositions adapted for oral administration may beprovided as capsules or tablets; as powders or granules; as solutions,syrups or suspensions (in aqueous or non-aqueous liquids); as ediblefoams or whips; or as emulsions. Tablets or hard gelatine capsules maycomprise lactose, starch or derivatives thereof, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, stearic acid or saltsthereof. Soft gelatine capsules may comprise vegetable oils, waxes,fats, semi-solid, or liquid polyols etc. Solutions and syrups maycomprise water, polyols and sugars.

An active agent intended for oral administration may be coated with oradmixed with a material that delays disintegration and/or absorption ofthe active agent in the gastrointestinal tract (e.g., glycerylmonostearate or glyceryl distearate may be used). Thus, the sustainedrelease of an active agent may be achieved over many hours and, ifnecessary, the active agent can be protected from being degraded withinthe stomach. Pharmaceutical compositions for oral administration may beformulated to facilitate release of an active agent at a particulargastrointestinal location due to specific pH or enzymatic conditions.

Pharmaceutical compositions adapted for transdermal administration maybe provided as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time.Pharmaceutical compositions adapted for topical administration may beprovided as ointments, creams, suspensions, lotions, powders, solutions,pastes, gels, sprays, aerosols or oils. For topical administration tothe skin, mouth, eye or other external tissues a topical ointment orcream is preferably used. When formulated in an ointment, the activeingredient may be employed with either a paraffinic or a water-miscibleointment base. Alternatively, the active ingredient may be formulated ina cream with an oil-in-water base or a water-in-oil base. Pharmaceuticalcompositions adapted for topical administration to the eye include eyedrops. In these compositions, the active ingredient can be dissolved orsuspended in a suitable carrier, e.g., in an aqueous solvent.Pharmaceutical compositions adapted for topical administration in themouth include lozenges, pastilles and mouthwashes.

Pharmaceutical compositions adapted for nasal administration maycomprise solid carriers such as powders (preferably having a particlesize in the range of 20 to 500 microns). Powders can be administered inthe manner in which snuff is taken, i.e., by rapid inhalation throughthe nose from a container of powder held close to the nose.Alternatively, compositions adopted for nasal administration maycomprise liquid carriers, e.g., nasal sprays or nasal drops. Thesecompositions may comprise aqueous or oil solutions of the activeingredient. Compositions for administration by inhalation may besupplied in specially adapted devices including, but not limited to,pressurized aerosols, nebulizers or insufflators, which can beconstructed so as to provide predetermined dosages of the activeingredient. In a preferred embodiment, pharmaceutical compositions ofthe invention are administered via the nasal cavity to the lungs.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injectable solutions orsuspensions, which may contain antioxidants, buffers, bacteriostats andsolutes that render the compositions substantially isotonic with theblood of an intended recipient. Other components that may be present insuch compositions include water, alcohols, polyols, glycerine andvegetable oils, for example. Compositions adapted for parenteraladministration may be presented in unit-dose or multi-dose containers,for example sealed ampules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of asterile liquid carrier, e.g., sterile saline solution for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tablets.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lidocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water-free concentrate in a hermetically-sealedcontainer such as an ampule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampule of sterile saline can be providedso that the ingredients may be mixed prior to administration.

In another embodiment, for example, a drug, such as the C5a inhibitordescribed herein, can be delivered in a controlled-release system. Forexample, the inhibitor may be administered using intravenous infusion,an implantable osmotic pump, a transdermal patch, liposomes, or othermodes of administration. In one embodiment, a pump may be used (seeSefton (1987) CRC Crit. Ref Biomed. Eng. 14: 201-240; Buchwald et al.(1980) Surgery 88:507-516; Saudek et al. (1989) N. Eng. J. Med.321:574-579). In another embodiment, the compound can be delivered in avesicle, in particular a liposome (see R. Langer (1990) Science249:1527-1533; Treat et al. (1989) in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,N.Y., 353-365; WO 91/04014; U.S. Pat. No. 4,704,355). In anotherembodiment, polymeric materials can be used (see Medical Applications ofControlled Release (1974) Langer and Wise (eds.), CRC Press: Boca Raton,Fla.; Controlled Drug Bioavailability, Drug Product Design andPerformance, (1984) Smolen and Ball (eds.), Wiley: N.Y.; Ranger andPeppas (1953) J. Macromol. Sci. Rev. Macromol. Chem. 23: 61; see alsoLevy et al. (1985) Science 228:190; During et al. (1989) Ann. Neurol.25: 351; Howard et al. (1989) J. Neurosurg. 71: 105).

In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the target cells, tissue ororgan, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson (1984) 115-138 in Medical Applications of Controlled Release,vol. 2). Other controlled release systems are discussed in the review byLanger (1990, Science 249: 1527-1533).

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions or the C5a inhibitors of the inventionlocally to the area in need of treatment; this may be achieved by, forexample, and not by way of limitation, local infusion during surgery,topical application, e.g., in conjunction with a wound dressing aftersurgery, by injection, by means of a catheter, by means of asuppository, or by means of an implant, said implant being of a porous,non-porous, or gelatinous material, including membranes, such assilastic membranes, or fibers.

Selection of the preferred effective dose will be determined by askilled artisan based upon considering several factors which will beknown to one of ordinary skill in the art. Such factors include theparticular form of the pharmaceutical composition, e.g. polypeptide orvector, and its pharmacokinetic parameters such as bioavailability,metabolism, half-life, etc., which will have been established during theusual development procedures typically employed in obtaining regulatoryapproval for a pharmaceutical compound. Further factors in consideringthe dose include the condition or disease to be prevented and/or treatedor the benefit to be achieved in a normal individual, the body mass ofthe patient, the patient's age, the route of administration, whetheradministration is acute or chronic, concomitant medications, and otherfactors well known to affect the efficacy of administered pharmaceuticalagents. Thus, the precise dosage should be decided according to thejudgment of the practitioner and each patient's circumstances, e.g.depending upon the condition and the immune status of the individualpatient, and according to standard clinical techniques.

The following figures and examples are merely illustrative of thepresent invention and should not be construed to limit the scope of theinvention as indicated by the appended claims in any way.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. IFX-1 biological features.

FIGS. 1A and 1B: Blocking activity of IFX-1 to human (FIG. 1A) andmonkey (FIG. 1B) eC5a was tested in ZAP-CD11b assay. The data arerepresentatives of 3 separate experiments using different donors.

FIG. 1C: IFX-1 concentrations were measured in the plasma samples fromthe monkeys 0, 1, 3, 5, and 7 days after infection and antibodyadministration (n=4 for day 0, 1, 3; n=2 for day 5, 7).

FIG. 2. Complement activation in AGM lungs after H7N9 virus infection.

FIGS. 2A, 2B and 2C: Quantitative RT-PCR analysis for C3aR (FIG. 2A),C5aR (FIG. 2B) and MASP2 (FIG. 2C) were performed on 18 (A/H7N9 group)and 6 (mock group) collected samples from all lung lobes at day 3post-infection. The data presented are the fold-change (Mean±SEM) ascompared to mock.

FIGS. 2D, 2E, and 2F: Concentrations of C3a (FIG. 2D), C5a (FIG. 2E) andC5b-9 (FIG. 2F) in A/H7N9-infected AGM plasma were measured byquantitative ELISA. Data are expressed as Mean±SEM on indicatedtime-point (n=6 at day 0, 1 and 3; n=3 at day 5). *** means P<0.001 vs.day 0.

FIG. 3. Alleviated ALI after A/H7N9 virus infection with anti-C5aantibody treatment.

FIG. 3A: Semiquantitative histopathological analysis at day 3 revealedalleviated lung damages in IFX-1-treated AGMs compared with that of AGMsreceiving A/H7N9 infection only (2 AGMs in IFX-1 treated group and 3AGMs for the control group at each time point).

FIG. 3B: Body temperature change (Mean±SEM) at indicated time-pointsfollowing A/H7N9 infection. Data shown were calculated by subtractingthe temperature measured at day 0 (6 AMGs in A/H7N9 group and 4 AMGs inA/H7N9+IFX-1 group at day 0 and day 3 post-infection; 3 and 2 AMGs leftin respective groups at day 5 and day 7). * means P<0.05 vs A/H7N9group.

FIG. 3C: Lung viral titer at day 3 post-infection was determined inhomogenized samples collected from all lung lobes (n=18 in A/H7N9 groupfrom three AMGs and n=12 in A/H7N9+IFX-1 group from two AMGs). Data wereexpressed as TCID₅₀ per gram of lung tissue (Mean±SEM), and dotted lineindicated the limit of detection.

FIG. 4. Reduced inflammatory responses in AGMs after H7N9 virusinfection with anti-C5a antibody treatment.

FIGS. 4A to 4F: Quantitative ELISAs were performed to measure theconcentrations of IL-1β (FIG. 4A), IL-6 (FIG. 4B), IP-10 (FIG. 4C),IFN-γ (FIG. 4D), TNF-α (FIG. 4E) and MCP-1 (FIG. 4F) in AGM serumsamples. Data presented are concentrations (Mean±SEM) of cytokine andchemokine at indicated time-point, n=6 in A/H7N9 group (solid circle)and n=4 in A/H7N9+IFX-1 group (open circle) at day 0, 1 and 3post-infection; 3 and 2 AMGs left in respective groups at day 5). * and*** mean P<0.05 and P<0.001 respectively vs. A/H7N9 group.

FIGS. 4G and 4H: Semiquantitative analysis of macrophage (FIG. 4G) andneutrophil (FIG. 4H) counts in lungs at day 3 post-infection (n=3 inA/H7N9 group and n=2 in A/H7N9+IFX-1 group).

EXAMPLES

1. Materials and Methods

1.1 Ethics Statement

All procedures involving animals were approved by the Laboratory AnimalCenter, State Key Laboratory of Pathogen and Biosecurity, BeijingInstitute of Microbiology and Epidemiology IACUC's (The permitted numberis BIME 2013-15). The study of animals was carried out in strictaccordance with the recommendations in the Guide for the Care and Use ofLaboratory Animals.

1.2 African Green Monkey Model of H7N9 Virus Infection

Twelve 2-4 years old young adult African Green Monkeys (AGMs) were usedin this study, and all the experiments handling live virus andbiological samples with a potential contamination of live virus wereperformed in the biosafety level 3 laboratory. After being anesthetizedby intraperitoneal (i.p.) injection of ketamine (5 mg/kg), ten AGMs wereinoculated with A/Anhui/1/2013 (H7N9) virus (10⁶TCID₅₀) intratracheallyand two AGMs were inoculated with the same volume of PBS intratracheallyas the negative control. Four of the ten AGMs inoculated with virus weretreated with anti-C5a monoclonal antibody (IFX-1; 5 mg/kg) intravenously30 min after virus inoculation (H7N9+anti-C5a Ab group). Six of the tenAGMs were treated with PBS as control (H7N9+PBS group). The samples fromthree monkeys in H7N9+PBS group and two monkeys in H7N9+anti-C5aantibody group were collected on days 3 and 7 after infection,respectively. The samples of normal controls were collected on day 3.Heparin plasma and serum were collected on days 0, 1, 3, 5, 7 from allthe animals and stored at −70° C. until analysis, and used forevaluating the levels of complement activation products or cytokines.

The animals were euthanized on day 3 after infection by exsanguinationunder ketamine anesthesia. After being anesthetized, the AGMs'temperature was monitored, and nasal and pharyngeal swabs were taken andplaced in 1 ml Dulbecco's modified Eagle's medium supplemented with 100IU penicillin/ml and 100 μg of streptomycin/ml. The swabs were stored at−70° C. until the TCID₅₀ analysis was performed. Necropsies wereperformed according to a standard protocol. For semiquantitativeassessment of gross pathology, the percentage of affected lung tissue atnecropsy from each lung lobe was calculated by the area of consolidationand dark red discoloration in each lobe to determine the gross pathologyscore. For reverse transcription (RT)-PCR, samples were stored in RNAstore liquid (Tiangen Biotech Co., Ltd) at 4° C. overnight and thenstored at −70° C. until RT-PCR analysis was performed. Forhistopathology study, the trachea, lung tissues from the cranial,medial, and caudal lobes, liver, spleen, kidney, intestine, brain andlymph nodes were suspended in 10% neutral-buffered formalin overnightand embedded in paraffin, cut at 4 μm and stained with hematoxylin andeosin (H&E) and used for immunohistochemistry.

1.3 Histopathologic Analysis of Lung Damage

Lungs were collected and sampled in a standard procedure from thecranial, medial, and caudal lobes of the lung. Sections of 4 μmthickness were stained with hematoxylin and eosin (H&E) and examined bylight microscopy. Trachea and bronchial lesions were assessed accordingto the extent of denaturated epithelials and inflammatory cellinfiltration in the submucous membrane. The injury of parenchyma wasanalyzed according to the denaturated epithelials, degeneration andnecrosis of alveoli pneumocytes, infiltration of inflammatory cells andexpansion of parenchymal wall, hemorrhage and interstitial edema (Sun,S. et al. 2011, Am J Respir Cell Mol Biol; 18:834-842). The cumulativescores of size and severity of degeneration or inflammation provided thetotal score per animal, and the average was taken as the total score forthat group.

1.4 Immunohistochemistry Staining for Macrophages and Neutrophils

Formalin-fixed, paraffin-embedded lung sections were de-paraffinizedwith xylene and hydrated using graded alcohols. The infiltration ofmacrophages, neutrophils and T lymphocytes was assessed using thefollowing antibodies: CD68, and Myeloperoxidase (MPO) (Beijing ZhongshanBiotechnology Co., Ltd., China). Antibodies were detected using astandard streptavidin-biotin detection system (Beijing ZhongshanBiotechnology Co., Ltd., Beijing, China) according to the manufacturer'sinstructions.

For semi-quantitative assessment of macrophage and neutrophilinfiltration, 30-50 arbitrarily chosen 40× objective fields of lungparenchyma in each lung section were examined by light microscopy forthe presence of macrophages or neutrophils in a blinded fashion. Thecumulative scores for each animal were expressed as the number ofpositive fields per 100 fields (%) (Sun, S. et al. 2013, supra).

1.5 Measurement of IFX-1 Concentrations and CD11b Assay

IFX-1 levels in the monkey plasma samples were measured by the standardenzyme-linked immunosorbent assay (ELISA) provided by InflaRx GmbH,Germany. CD11b assay was used to assess the blocking efficiency of IFX-1in human and monkey. Briefly, human or monkey blood was stimulated withplasma or zymosan-activated plasma (ZAP; containing endogenousC5a-eC5a). Different concentrations of IFX-1 and control human IgG4antibody (Sigma, WI, USA) were added in the assay to determine the IFX-1blocking activity without any pre-incubation of antibody and eC5a, andeC5a levels were measured by an ELISA kit provided by InflaRx GmbH,Germany. After stimulation, anti-mouse CD11b:FITC or isotype controlmAbs (BD Bioscience, NJ, USA) was added and incubated. Immediately afterthe lysing step of red blood cells, CD11b expression on the gatedgranulocytes was analyzed by the BD FACSCanto™ II flow cytometer. Meanfluorescence intensity (MFI) of FITC-labeled granulocytes was used toexamine the level of CD11b expression.

1.6 Measurement of Inflammatory Cytokines and C3a, C5a, C5b-9 in Plasma

Serum or plasma samples of infected AGMs were collected at indicatedtimes and stored at −70° C. before measurement. Cytokine levels ofIL-1β, IL-6, IFN-γ, TNF-α, MCP-1 and IP-10 were measured using themonkey ELISA kits from U-CyTech biosciences or Uscn life science Inc.C3a and C5b-9 levels were measured using the human ELISA kits from BDbiosciences, and C5a ELISA was provided by InflaRx GmbH, Jena, Germany.In brief, 100 μl of diluted AGMs serum or plasma were added to the platepre-coated with antibody specific for individual AGMs cytokines, MPO,C3a, C5a and C5b-9 and incubated at 4° C. overnight. Following a wash,enzyme-linked specific antibodies were added and incubated at 37° C. for1 hour. After washing the plates, substrate solution was added andincubated at 37° C. for 30 minutes. Assays were developed using TMB, andthe reaction was stopped by adding 1N H₂SO₄. The absorbance at 450 nmwas measured by an ELISA plate reader (Synergy 2, Bio Tek), and theamount of AGMs cytokines, or C3a, C5a and C5b-9 were determined by thestandard curve obtained in the measurement.

1.7 Detection of C3aR mRNA, C5aR mRNA and MASP2 Expression

Total RNA was isolated from lung tissue of the cranial, medial, andcaudal lobes of AGMs, and relative quantitative real-time RT-PCR wasperformed. The relative C3aR, C5aR and mannose-bindingprotein-associated serine protease 2 (MASP2) expression data wereanalyzed using the 2^(−ΔΔ) ^(C) T method (Livak, K. J. & Schmittgen T.D. 2001. Methods 25:402-408).

1.8 Virus Titers in Tissues

Bronchia and six parts of lung tissue samples from cranial, medial, andcaudal lobes of the right and left lung respectively in each infectedAGMs were harvested at indicated times and homogenized using OMNI BEADRUPTOR 24 tissue grinders (OMNI International, INC.) in minimalessential medium (MEM) plus antibiotics to achieve 10% (w/v)suspensions. Viral titers in tissues were determined by 50% tissueculture infective dose (TCID₅₀) as described (Zhao, G. et al. 2010.Virology Journal, 7:151-156). In brief, monolayers of MDCK cells wereinoculated with tenfold serial dilutions of homogenates of AGMs organsin quadruplicate. Two hours after inoculation, supernatants were removedand replaced with MEM plus antibiotics and 2 μg/ml TPCK-trypsin (Sigma).Following 3 days' observation of viral cytopathic effect (CPE),infection of the cells was indicated by hemagglutinating activity using0.5% turkey erythrocytes. Tissue viral titers were calculated by theReed and Muench method and expressed as Log₁₀TCID₅₀/g of tissues.

1.9 Statistical Analysis

Student's t-test with Welch's correction was used for the comparison ofdata in RT-PCR analysis, semiquantitative histopathological analysis,lung viral titer detection and semiquantitative analysis of macrophageand neutrophil counts. Data of plasma concentrations of C3a, C5a andC5b-9 and blocking activity of IFX-1 to human eC5a were compared usingone-way ANOVA with Dunnett's post-test. Differences in temperaturechanges and inflammatory cytokines and chemokines between the groups atindicated time-points were compared using two-way ANOVA with Bonferronipost-test. P values lower than 0.05 were considered statisticallysignificant. The data are represented as the mean±s.e.m. All analyseswere performed in Graphpad Prism version 5.01.

2. Results

2.1 IFX-1 and its Biological Activities

IFX-1 is a chimeric human/mouse monoclonal IgG4 antibody developed byInflaRx GmbH, Germany. The antibody is an IgG4 kappa antibody producedby CHO (Chinese Hamster Ovary) cell line and consists of murine heavyand kappa light chain variable (VH and VL) regions and human gamma 4heavy chain and kappa light chain constant regions. As disclosed byInflaRx GmbH, IFX-1 has been tested in a monkey toxicological study andin a human phase I trial, and has demonstrated a good safety profile forfurther clinical trials.

IFX-1 blocking activity was tested by CD11b assay using endogenous C5a(eC5a) generated from human ZAP samples from 8 different donors. Asshown in FIG. 1A, IFX-1 significantly decreased the CD11b expression onhuman granulocytes by over 80% at an Ab:Ag molar ratio of 0.5:1. Theblocking activity of IFX-1 on eC5a-induced CD11b up-regulation reachedup to 98% when Ab:Ag ratios of 1:1 and 2:1 were applied.

Blocking activity to the monkey C5a was also tested by ZAP-CD11b assayusing monkey ZAP and monkey whole blood. IFX-1 is capable of blockingthe ZAP-driven CD11b upregulation on monkey granulocytes by 100% (FIG.1B), indicating that IFX-1 is a fully functional blocking antibody tomonkey eC5a.

In the monkey model of H7N9 virus infection, a dose of 5 mg/kg of IFX-1was applied intravenously to treat the four infected monkeys, and itslevels were then monitored by the pharmacokinetics assay. There wasapproximately 40 μg/ml of IFX-1 found in the four treated monkeys 1 dayafter the treatment, and the level of IFX-1 dropped to around 10 μg/mlin the two monkeys left at day 7 (FIG. 1C). These data indicate thatIFX-1 is fully functional to block monkey eC5a and the dose applied forthe treatment is sufficient in the model.

2.2 The Pathogenesis of H7N9 Virus-Infected African Green Monkey (AGM)

To explore the pathogenesis of H7N9 virus and the host immune responsesto the virus infection, eight AGMs were infected with H7N9 virus(AH1/H7N9), and two AGMs were treated with Mock. Three animals wereeuthanized on day 3, three further animals were euthanized on day 7 andthe remaining two animals were euthanized on day 14 after infection.Lung, trachea, heart, liver, kidney, spleen, brain and intestine weretaken from H7N9 virus-infected AGMs and homogenized for virusisolations. Virus can be isolated from lung and trachea but not fromother tissue homogenates on day 3 post-infection (Table 4). On day 7 and14, no virus could be isolated from any of the collected tissues. AGMserum hemagglutination inhibition (HI) titers were detected 7 days afterinfection, and the HI titers on day 14 were 1:80 to 1:160 (Table 4).Virological and serological tests proved that AGMs were effectivelyinfected by H7N9 virus.

TABLE 4 Tissue virus isolation and serum HI titer. Group A/H7N9 MockDays post-infection 3 d 7 d 14 d 3 d 7 d AGM Number 1 2 3 4 5 6 7 8 1 2Virus Lung + + + − − − − − − − isolation Trachea + + + − − − − − − −Heart − − − − − − − − − − Liver − − − − − − − − − − Kidney − − − − − − −− − − Spleen − − − − − − − − − − Brain − − − − − − − − − − Intestine − −− − − − − − − − Serum HI titers <1:10 <1:10 <1:10 1:10 1:20 1:20 1:801:160 <1:10 <1:10

On further microscopic observation (data not shown), the peak damage oflung was observed on day 3 after H7N9 virus infection and recoveredgradually afterwards. On day 3, when compared with the control group,which was inoculated with PBS, a multifocal trachea-brochoadenitis wasobserved in the infected lungs, and it was characterized by thedenaturated epithelials and moderate infiltration of lymphocytes,macrophages, neutrophils and occasional eosinophils in the submucousmembrane of the trachea. The lesions found in the lung infected withH7N9 virus were acute exudative diffuse pulmonary damage which wascharacterized by denaturated and collapsed epithelials in bronchiolesand terminal bronchioles, diffused and thickened alveolar septa withinfiltration of lymphocytes, macrophage and neutrophils, denaturated andcollapsed pneumocytes, and multifocal hemorrhage in lung interstitial,exudates and severe edema. The endothelial were denaturated with a largenumber of inflammatory cells adherence and damaged basement membrane.The ultrastructure observation showed degenerated pulmonary epithelialcells with swelling mitochondrial and denatured endoplasmic reticulum,damaged blood-gas barrier with cell debris falling off to alveolar spaceand inflammatory cells infiltration in the interstitial edema (data notshown).

Among all the other organs examined, including brain, heart, intestine,spleen and kidney, spleen was the only organ showing a certain level ofhistopathological changes after H7N9 viral infection with a modestelevated number of phagocytes in red pulp and artery cuff of spleen aswell as hyperemia and focal haemorrhage in red pulp (data not shown).There was no injury found in other organs such as brain, intestine andkidney.

2.3 Complement Activation in Lung after H7N9 Virus Infection

Although complement activation plays a pivotal role in defense againstpathogen invasion, accumulated studies demonstrated that excessivecomplement activation was associated with a variety of autoimmune andinflammatory diseases (Klos, A. et al. 2009, supra). In this study, thereal-time RT-PCR analysis revealed that the transcriptional levels ofC3aR, C5aR and MASP2 expression were significantly upregulated at day 1of H7N9 virus infection (FIGS. 2A, B and C). In addition, the majorcomplement activation product C3a, C5a and C5b-9 levels in plasmasamples from AGMs were markedly elevated after H7N9 virus infection. C3aand C5b-9 maintained the high levels at all time-points from 1 to 5days, while the C5a level was significantly increased at day 1 and day 3and returned close to the background level at day 5 (FIGS. 2D, E and F).Furthermore, the enhanced protein expression of C3aR and C5aR in lungsections especially in the bronchiole epithelials and the tissues withsevere inflammation were demonstrated by immunohistochemistry stainingafter H7N9 virus infection (data not shown). C3c staining, anotherindicator of complement activation, also showed an increased depositionin AGM lungs 3 day after H7N9 virus infection (data not shown). Thesedata indicated that the complement system is extensively activated inthe circulation as well as in the lungs after H7N9 infection.

2.4 Anti-C5a Antibody Treatment Alleviated the Clinical Signs of ALIInduced by H7N9 Virus Infection

To investigate the role of complement activation in the pathogenesis ofH7N9 virus infection induced-lung injury, anti-C5a antibody IFX-1 (5mg/kg) was intravenously injected in the infected AGMs immediately aftervirus infection. Gross pathology revealed that the infected lungs ofAGMs presented multifocal consolidation and dark red discoloration whichwas most prevalent on the dorsal surface of lungs, while the lungs fromthe infected AGMs treated with anti-C5a presented almost normalappearance with very little dark red discoloration (data not shown).Histopathological analysis demonstrated that all the infected AGMsdeveloped some degree of pulmonary damage with mild or multifocalbronchointerstitial pneumonia. However, the lung pathology is clearlyimproved in the lungs from infected AGMs with anti-C5a treatment. On day3 after infection, the untreated AGMs showed large and multifocal lunglesions with desquamation of bronchiolar epithelial cells, degenerationand necrosis of alveolar epithelial, abundant interstitial edema,multifocal hemorrhage and strong inflammatory infiltration, while mildto moderate expansion of parenchymal wall with less interstitial edema,a much lower number of inflammatory cell infiltrates and a clearly lowerdegree of lung damage are presented in the infected AGMs with anti-C5atreatment (data not shown). Although the lung injury appeared to belighter on day 7 than that on day 3 in both the treated and nontreatedgroups, the degeneration of bronchiolar epithelial cells andpneumocytes, the edema of interstitial especially around the bloodvessels were more severe in untreated AGMs than those in treated AGMs onday 7 (data not shown). The semiquantitative histological analysis forthe AGM lungs obtained 3 days after infection indicated that the lunghistopathologic injury score was greatly attenuated by IFX-1 treatment(FIG. 3A).

The AGMs treated with IFX-1 antibody showed a smaller body temperaturefluctuation after H7N9 virus infection when compared to that ofnon-treated AGMs (FIG. 3B). Surprisingly, the results of viral titers inhomogenized lung tissues showed that the mean lung viral titer intreated AGMs was approximately 1.8 log lower than that in untreated AGMs(p<0.01) (FIG. 3C), which indicated that the virus replication wassomehow reduced after anti-C5a antibody treatment.

2.5 Anti-C5a Antibody Treatment Reduced Inflammatory Responses Initiatedby H7N9 Virus Infection in AGMs

To further determine the role of complement activation on inflammatoryresponses initiated by H7N9 virus infection in AGMs, the inflammatorycytokines and chemokines as well as the infiltration of macrophages andneutrophils were evaluated in H7N9 virus infected AGMs with or withoutthe IFX-1 antibody treatment. As shown in FIG. 4, all the inflammatorymediators investigated in the study were significantly elevated afterinfection. IL-1β, IP-10 and MCP-1 reached to the peak expression asearly as 1 day after infection, while IL-6, TNF-α and IFN-γ showed thepeak expression at day 3, and the levels of all the mediators weredeclined at day 5. The overall expression levels of these inflammatorymediators appear to be significantly hindered in the anti-C5a treatedmonkeys.

The infiltration of macrophages and neutrophils was measured byimmunohistochemistry staining of CD68 and MPO expression in lung tissuesections of AGMs prepared at day 3 post-infection (data not shown). Thedata showed that both neutrophils and macrophages increased markedly inthe infected lungs and the degree of infiltration had a positivecorrelation with the lesion of lungs. However, the numbers ofinflammatory infiltrates especially neutrophils decreased significantlyin the lungs of IFX-1 treated monkeys when compared with the non-treatedAGMs (FIG. 4G, H). Collectively, the data confirmed the effectivetherapeutic effect of anti-C5a treatment on the ALI and systemicinflammation initiated by H7N9 viral infection.

Sequence Listing Free Text Information

SEQ ID NO: 6 IFX-1 CDR3 heavy chain SEQ ID NO: 7 INab708 CDR3 heavychain SEQ ID NO: 8 IFX-1 CDR3 light chain SEQ ID NO: 9 INab708 CDR3light chain SEQ ID NO: 10 IFX-1 CDR2 heavy chain SEQ ID NO: 11 INab708CDR2 heavy chain SEQ ID NO: 12 IFX-1 CDR2 light chain SEQ ID NO: 13INab708 CDR2 light chain SEQ ID NO: 14 IFX-1 CDR1 heavy chain SEQ ID NO:15 INab708 CDR1 heavy chain SEQ ID NO: 16 IFX-1 CDR1 light chain SEQ IDNO: 17 INab708 CDR1 light chain SEQ ID NO: 18 IFX-1 FR1 heavy chain SEQID NO: 19 IFX-1 FR2 heavy chain SEQ ID NO: 20 IFX-1 FR3 heavy chain SEQID NO: 21 IFX-1 FR4 heavy chain SEQ ID NO: 22 IFX-1 FR1 light chain SEQID NO: 23 IFX-1 FR2 light chain SEQ ID NO: 24 IFX-1 FR3 light chain SEQID NO: 25 IFX-1 FR4 light chain SEQ ID NO: 26 INab708 FR1 heavy chainSEQ ID NO: 27 INab708 FR2 heavy chain SEQ ID NO: 28 INab708 FR3 heavychain SEQ ID NO: 29 INab708 FR4 heavy chain SEQ ID NO: 30 INab708 FR1light chain SEQ ID NO: 31 INab708 FR2 light chain SEQ ID NO: 32 INab708FR3 light chain SEQ ID NO: 33 INab708 FR4 light chain

The invention claimed is:
 1. A method for the reduction of viral load ina subject suffering from viral pneumonia caused by an HxNx influenzavirus, said method comprising the step of: administering a therapeuticamount of an inhibitor of C5a to said subject, thereby reducing viralload in said subject, wherein the inhibitor of C5a is a binding moietyspecifically binding to human C5a, wherein said binding moietyspecifically binds to a conformational epitope formed by amino acidsequences NDETCEQRA (SEQ ID NO: 2) and SHKDMQL (SEQ ID NO: 3) of humanC5a, wherein the binding moiety binds to at least one amino acid withinthe amino acid sequence according to SEQ ID NO: 2 and to at least oneamino acid within the amino acid sequence according to SEQ ID NO: 3, andwherein said binding moiety is an antibody or an antigen-bindingfragment thereof.
 2. The method according to claim 1, wherein the HxNxinfluenza virus is selected from the group consisting of H1N1, H1N3,H2N2, H3N2, H5N1, H7N2, H7N3, H7N7, H7N9, H9N2, H10N7, and H10N8.
 3. Themethod according to claim 1, wherein the subject is a human.
 4. Themethod according to claim 1, wherein said binding moiety is an antibodyor an antigen-binding fragment thereof, wherein said antibody orantigen-binding fragment thereof comprises (i) a heavy chain CDR3sequence as set forth in SEQ ID NO: 6; or (ii) a heavy chain CDR3sequence as set forth in SEQ ID NO:
 7. 5. The method according to claim1, wherein said binding moiety is an antibody or an antigen-bindingfragment thereof, wherein said antibody or antigen-binding fragmentthereof comprises (iii) a light chain CDR3 sequence as set forth in SEQID NO: 8; or (iv) a light chain CDR3 sequence as set forth in SEQ ID NO:9.
 6. The method according to claim 1, wherein said binding moiety is anantibody or an antigen-binding fragment thereof, wherein said antibodyor antigen-binding fragment thereof comprises at least one of thefollowing sequences: (v) a heavy chain CDR2 sequence according to SEQ IDNO: 10; (vi) a heavy chain CDR2 sequence according to SEQ ID NO: 11;(vii) a light chain CDR2 sequence according to SEQ ID NO: 12; (viii) alight chain CDR2 sequence according to SEQ ID NO: 13; (ix) a heavy chainCDR1 sequence according to SEQ ID NO: 14; (x) a heavy chain CDR1sequence according to SEQ ID NO: 15; (xi) a light chain CDR1 sequenceaccording to SEQ ID NO: 16; or (xii) a light chain CDR1 sequenceaccording to SEQ ID NO: 17.